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IIIIIIIIIIIIIIIIIIIIIIIIIIII \ljlllllllIlllllllllljlllIlllllll “ THESIS \ ‘ 9 020 2 ’5 ll This is to certify that the dissertation entitled Acfidafiod at QhogpMoLigasz. A2, 31 o\ chlovinocred biwems (mes) Ma omev ? ‘l QNoxith-ted Comeognas presented by 11305 (rodeo O\2ve(o—\lexbe\ has been accepted towards fulfillment of the requirements for E ll D. degree in ”M: Van tidlajciand Bx/ca/csc/ and {nu/iron mental Toxins/(5‘, Date [0111 Z! i? MSU is an Affirmative Anion/Equal Oppnriunily Institution 0‘ 12771 LEBRARY Michigan State ' University J M —— ACTIVATION OF PHOSPHOLIPASE A2 BY POLYCHLORINATED BIPHENYLS (PCBs) AND OTHER CHLORINATED COMPOUNDS By JESUS TADEO OLIVERO VERBEL A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree Of DOCTOR OF PHILOSOPHY Department Of Pharmacology and Toxicology Institute for Environmental Toxicology 1999 ABSTRACT ACTIVATION OF PHOSPHOLIPASE A; BY POLYCHLORINATED BIPHENYLS (PCBs) AND OTHER CHLORINATED COMPOUNDS BY JESUS OLIVERO VERBEL Neutrophils are immune cells that constitute the first line Of defense against pathogens. Upon stimulation these cells undergo biochemical processes Of importance in host defense and inflammation. One group Of chemicals that stimulates neutrophils is the polychlorinated biphenyls (P083). PCBs are compounds Of environmental concern because they are present in all the ecosystems and have a broad spectrum Of biological effects. An important event during neutrophil stimulation by P085 is the activation Of phospholipase A2 (PLAz), an enzyme responsible for the release Of arachidonic acid from cell membranes and required for degranulation and superoxide anion production, two Of the major cellular responses associated with neutrophil activation. This study investigated the cellular and molecular mechanisms controlling the activation Of PLA2 by PCBs. On‘hO-PCBs but not non-orthO-PCBs induced increases in intracellular Ca2+ concentration, phosphorylation Of p44 MAPK and activation Of two isoforms Of PLA2, a Cay-dependent (cPLAz) and a Ca”- independent PLA2 (iPLAz). Changes in Ca2+ homeostasis and activation Of iPLAz were independent events. PCBs targeted the same intracellular store as, and blocked Ca2+ influx induced by, the chemotactic agent formyl- methyOnyI-leucyl-phenylalanine (fMLP). Pharmacological intervention Of the PCB signalling suggested that activation Of PLA2 occurs through transduction pathways involving tyrosine kinases, protein kinase C, mitogen activated protein kinase and Ca2+lcalmodulin. Activation Of PLAZ was also triggered by a variety Of other organochlorine compounds that share a particular substructure similar tO that found in on‘hO-PCBs. This motif, called the 0G motif, was present in on‘hO-PCBs, (1-, 5-, and y- hexachlorocyclohexane (HCCH), dieldrin and chlordane, and was absent in inactive organochlorines such as [3—HCCH and non-orthO-PCBs. It consists Of a planar, hydrophobic structure linked tO a perpendicular, negatively charged atom through a rigid bridge. Copyright by Jesus OliverO-Verbel 1999 TO my parents, Rafael y Carmen to my lovely wife Isabel to my children, Maria and Catalina to the honest people from my country ACKNOWLEDGMENTS I would love to give thanks to the following people and institutions: Dr. Patricia Ganey, my professor and mentor My thesis committee: Dr. Robert Roth Dr. Lawrence Fischer Dr. Norbert Kaminski Dr. B.V. Madhukar Lab People: John Buchweitz, Bryan Copple, Charles Barton, Eva Barton, Steve Yee, Shawn Kinser, Manju Thabolingam, Ali Mahajerin , Jamie Morgan, Sara Jehl and Oliva Primera. The Department Of Pharmacology and Toxicology and the Institute Of Environmental Toxicology Of Michigan State University. Colciencias, Fulbright and Laspau Universidad de Cartagena Republic Of Colombia My parents, my brother, my wife and my children vi TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES Chapter I INTRODUCTION I.1. Preface |.2. Neutrophils: Physiological function l.3. Neutrophils: Mechanisms Of activation I. 3.A. Calcium I. 3.8. Phospholipase C I. 3.C. Tyrosine kinases I. 3D. Protein Kinase C I. BE. Phospholipase D I. 3.F. Summary I. 4. Phospholipase A2 and cellular function. I. 5. Polychlorinated biphenyls I. 5.A. Structure I. 5.8. Sources, distribution and occurrence I. 5.0. ToxicOkinetics Of PCBs I. 5.0. Toxicology Of PCBs I. 5. Di. Toxicology Of dioxin-like PCBs I. 5. D.ii. Toxicology Of non-dioxin like PCBs I.5.E. On‘hO-chlorinated PCBs and neutrophil funcfion I.6. Cell activation by other chlorinated chemicals I.7. Summary Chapterll REGULATION OF CALCIUM HOMEOSTASIS BY POLYCHLORINATED BIPHENYLS IN RAT NEUTROPHILS: EFFECTS ON REPONSES TO THE CHEMOTACTIC AGENT FMLP ”.1 Summary vii xi xii (DCOOTU‘IOONA 11 12 12 17 17 17 20 20 22 23 26 27 30 32 33 “.2. Introduction ”.3. Materials and methods II.3.A. Chemicals II.3.B. Isolation Of rat, peritoneal neutrophils II.3.C. Measurement Of [032*], in rat neutrophils II.3.D. Statistical analysis ”.4. Results I|.4.A. Aroclor 1242-stimulated increases in [Ca2+], in rat neutrophils are both time- and dose-dependent II.4.B. OrthO-chlorinated but not non-ortho- chlorinated PCBs reproduce the effects Of Aroclor 1242 I|.4.C. Aroclor 1242 interferes with fMLP-induced increases in [Caz"]i. ”.5. Discussion Chapter III ROLE OF PROTEIN PHOSPHORYLATION IN ACTIVATION OF PHOSPHOLIPASE A2 BY THE POLYCHLORINATED BIPHENYL MIXTURE AROCLOR 124254 |lI.1. Summary III.2. Introduction III.3. Materials and methods III. 3.A. Chemicals III. 3.8. Isolation Of rat peritoneal neutrophils III. 3.C. Exposure tO PCBs_ III. 3.0. Determination Of PLA2 activity III. 3E. Detection Of phosphorylated p42/p44 MAPKs III. 3.F. Cytotoxicity assay III. 3.G. Statistical analysis II|.4. RESULTS lll.4.A. Aroclor 1242 induced release of [3H]-AA from neutrophils II.4.B. Involvement Of protein kinase pathways in Aroclor 1242-induced activation Of PLA2 III.5. Discussion Chapter IV CALCIUM/CALMODULlN-DEPENDENT REGULATION OF PHOSPHOLIPASE A2 DURING NEUTROPHIL ACTIVATION BY POLYCHLORINATED BIPHENYLS viii 34 35 35 36 36 38 38 38 42 42 47 55 56 59 59 59 6O 6O 61 62 62 63 63 65 72 78 IV.1. Summary IV.2. Introduction IV.3. Materials and methods IV.3.A. Chemicals IV.3.B. Isolation Of rat peritoneal neutrophils IV.3.C. Exposure to PCBs IV.3.D. Determination Of PLA2 activity IV.3.E. Neutrophil degranulation IV.3.F. Intracellular Free Ca2+ Measurements IV.3.G. Cytotoxicity assay IV.3.H. Statistical methods IV.4. Results IV.4.A. Involvement Of Ca2+lcalmodulin in PCB-induced activation Of PLA2 IV.4.B. TFP and Aroclor 1242-induced changes in neutrophil function IV.5. Discussion Chapter V BIOCHEMICAL SIGNALS INVOLVED IN THE ACTIVATION OF PLA2 BY 2,2’,4,4’-TETRACHLOROB|PHENYL V.1. Summary V.2. Introduction v.3. Materials and methods V.3.A. Chemicals V.3.B. Isolation Of rat peritoneal neutrophils V.3.C. Exposure to PCBs V.3.D. Determination Of PLA2 activity V.3.E. Detection Of phosphorylated cPLAz and p42/p44 MAPKs V.3.F . Cytotoxicity assay V.3.G. Statistical Methods V.4. Results V.4.A. Activation Of PLA2 by 2,2’,4,4’- tetrachlorbiphenyl and 3,3’,4,4’-tetrachlorobiphenyl V.4.B. Role Of protein kinase pathways and calmodulin in 2,2’,4,4’-tetrachlorobiphenyl- induced activation Of PLA2 V.4.C. Effects Of 2,2’,4,4’-tetrachlorobiphenyl and 3,3’,4,4’-tetrachlorobiphenyl on cPLA2 and p42/p44 MAPK phosphorylation 79 80 82 82 83 83 83 84 85 86 86 87 87 89 93 99 100 101 102 102 102 102 103 104 105 105 106 106 109 114 v.5. Discussion Chapter VI STRUCTURE-ACTIVITY RELATIONSHIPS FOR THE ACTIVATION OF RAT NEUTROPHIL PHOSPHOLIPASE A2 BY ORGANOCHLORINE COMPOUNDS V|.1. Summary VI.2. Introduction VI.3. Materials and methods VI.3.A. Chemicals VI.3.B. Isolation Of rat peritoneal neutrophils VI.3.C. Exposure to organochlorine compounds VI.3.D. Determination Of PLA2 activity VI.3.E. Cytotoxicity assay VI.3.F. Structure-activity relationships VI.3.F.i Data set VI.3.F.ii. Computational details Vl.3.F.iii. Model construction VI.4. Results VI.4.1. PLA2 activity induced by organochlorine compounds VI.4.2. Structure-activity relationships VI.5. Discussion Chapter VII SUMMARY BIBLIOGRAPHY 118 124 125 126 127 127 128 128 129 129 130 130 130 132 135 135 143 151 158 164 LIST OF TABLES Table II. Selected properties Of Phospholipases A2. 14 Table “.1. Net changes in basal [Ca2+] (nM) in rat neutrophils exposed to different concentrations Of Aroclor 1242. 40 Table IV.1. TMB-induced inhibition Of Aroclor 1242-stimulated release Of [3H]-AA from rat neutrophils. 88 Table VI. 1. Molecular descriptors for OCs in the Data set. 145 xi Figure l.1. Figure |.2. Figure ”.1. Figure ”.2. Figure ”.3. Figure ”.4. Figure ”.5. Figure ”.6. Figure |||.1. Figure |l|.2. Figure |ll.3. LIST OF FIGURES Signal transduction during neutrophil activation by fMLP Molecular structure Of polychlorinated biphenyls. Effect Of 10 ug/mL Aroclor 1242 on [CaZ+]i in rat neutrophils. Effect Of EGTA on Aroclor 1242- and fMLP- induced increase on [Ca2+], in rat neutrophils. Effects Of 2,4’-dich|orobiphenyl and 3,3’,4,4’- tetrachlorobiphenyl on [Ca2+], in rat neutrophils. Effects Of simultaneous addition Of fMLP and Aroclor 1242 on [Ca2+], in rat neutrophils. Effect Of fMLP applied at different times after exposure tO Aroclor 1242 on [Ca2+], in rat neutrophils. Effect Of Aroclor 1242 applied two minutes after exposure to fMLP on [Ca2+], in rat neutrophils. A. Time-course for Aroclor 1242-induced release Of [3H]-AA from labeled neutrophils. B. Dose- response curve for BEL-induced inhibition Of Aroclor 1242- elicited release Of [3H]-AA. Dose-response curve for inhibition Of Aroclor 1242 (10 ug/mL)-induced release Of [3H]-AA by Genistein or Daidzein. A. Dose-response curves for (A) SB 203580- and (B) PD 98059-induced inhibition Of Aroclor xii Pag 13 18 39 41 43 44 46 48 64 66 Figure III.4. Figure III.5. Figure IV.1. Figure |V.2. Figure lV.3. Figure V.1. Figure v.2. Figure V.3. Figure V.4. Figure V.5. Figure v.6. Figure V7. 1242 (10 ug/mL)-stimu|ated release Of [3H]-AA. Aroclor 1242-stimulated MAPK phosphorylation in rat neutrophils and effects Of BEL and PD 98059. Dose-response curves for (A) Staurosporine- and (B) RO 32-O432-induced inhibition Of Aroclor 1242 (10 ug/mL)-stimulated release Of [3H]-AA. Effects Of BEL on 10 ug/mL Aroclor 1242- induced increase in [Ca2+]i in rat neutrophils. Effects Of calmodulin inhibitors on phospholipase A2 activity induced by 10 pg/mL Aroclor 1242. Inhibitory effect Of (A) Aroclor 1242 and (B) Trifluoperazine onfMLP-induced neutrophil degranulation. Effects Of 2,2’4,4’-tetrachIorobiphenyl on neutrophil PLA2 activity and cytotoxicity. Effects Of 3,3’4,4’-tetrachIorobiphenyl on neutrophil PLA2 activity and cytotoxicity. Dose-response curve for inhibition Of 2,2’,4,4’- tetrachlorobiphenyl (25 uM)—induced release Of [3H]-AA by genistein. Dose-response curves for inhibition Of 2,2’,4,4’- tetrachlorobiphenyl (25 uM)-induced release Of [3H]-AA by PD 98059 and UO126. Dose-response curve for inhibition Of 2,2’,4,4’- tetrachlorobiphenyl (25 uM)-induced release Of [3H]-AA by RO-32-O432. Dose-response curve for inhibition Of 2,2’,4,4’- tetrachlorobiphenyl (25 uM)-induced release Of [3H]-AA by manumycin. Effects Of calmodulin inhibitors on 2,2’,4,4’- tetrachIorObiphenyI-induced PLA2 activity. xiii 68 69 71 90 91 92 107 108 110 111 112 113 115 Figure V.8. 2,2’,4,4’-tetrachlorobiphenyl but not 3,3’,4,4’- tetrachlorobiphenyl phosphorylates cPLA2, Figure V.9. p42/p44 MAPK phosphorylation response by PCBs: Effects Of genistein and PD 98059 on 2,2’4,4’- tetrachIorobiphenyl-induced phosphorylation Of p42/p44 MAPK. Figure VI.1. Molecular structure Of organochlorine compounds used in this study. Figure VI.2. General scheme for the development Of structure-activity relationships for organochlorine compounds and activation Of rat PLA2. Figure VI.3. Effects Of Ot-HCCH on neutrophil PLA2 activity and cytotoxicity. Figure VI.4. Effects Of 8-HCCH on neutrophil PLA2 activity and cytotoxicity. Figure VI.5. Effects Of gamma-HCCH on neutrophil PLA2 activity and cytotoxicity. Figure Vl.6. Effects Of B-HCCH on neutrophil PLA2 activity and cytotoxicity. Figure VI.7. Effects Of DDT on neutrophil phospholipase A; activity and cytotoxicity. Figure VI.8. Effect Of Dieldrin on neutrophil PLA2 activity and cytotoxicity. Figure VI.9. Effects Of Chlordane on neutrophil PLA2 activity and cytotoxicity. Figure Vl.10. Effects Of BEL on PLA2 activity induced by OC compounds. Figure V|.11. Molecular structures Of superimposed organochlorine compounds. xiv 116 117 131 134 136 137 138 139 140 141 142 144 147 Figure Vl.12. Figure Vl.13. Figure Vl.14. Figure Vl.15. Molecular structures of superimposed organochlorine compounds. Molecular structures Of superimposed organochlorine compounds. Superposition Of 2,2’,4,4’-tetrachlorobiphenyl and BEL, an inhibitor Of Cay-independent PLA2. Representation Of the OG motif Of OC compounds interacting with the toxicophore. XV 148 149 150. 154 [3H]-AA 12-HETE 12-HPETE AA AHH ANOVA BEL Ca2+ IC32+ii DAG DMF EROD fMLP HCCH HXAg iPLA2 LTB4 MAP K MP0 00 PCB PI LIST OF ABBREVIATIONS [3H]-arachidonic acid 12-hydroxyeicosa (5Z, 8Z, 10E, 14Z) tetraenoic acid 12-hydroperoxyeicosa (5Z, 8Z, 10E, 14Z) tetraenoic acid Arachidonic acid Aryl hydrocarbon hydroxylase Analysis Of variance Bromoenol Iactone Calcium Intracellular free Ca2+ concentration 1 ,2-sn-diacylgycerol N,N-Dimethylformamide 7-ethoxyresorufln-O-deethylase formyl-methionyI-IeucyI-phenylalanine Hexachlorocyclohexane Hepoxilin A8 lnositOI-1,4,5-trisphosphate Calcium-independent PLA2 Leukotriene B4 Mitogen-activated protein kinase Myeloperoxidase Organochlorine compound Polychlorinated Biphenyl Phosphoinositide xvi PWQ PKC PLA2 PLC PLD RyRs TCDD TFP TK PhophotidylinositOI-4,5-bisphosphate Protein Kinase C Phospholipase A2 Phospholipase C Phospholipase D RyanOdine receptors 2,3,7,8-tetrachlorodibenzo-p-dioxin Trifluoroperazine Tyrosine Kinase xvii Chapter I INTRODUCTION I.1. Preface Neutrophils are one Of the most important cells in the innate immune system. They are critical in host defense and inflammation. They perform a complex variety Of biochemical processes that involve different signaling cascades and lead tO the production Of oxidants and the release Of enzymes which ultimately battle pathogens. Polychlorinated biphenyls (PCBs), as well as other organochlorine compounds (OCs), are globally distributed in all Of the ecosystems and have been shown tO induce biochemical and functional changes in the neutrophil. The study Of the mechanisms by which PCBs or OCs affect neutrophils is important to determine the risk Of immune system impairment in people exposed tO these chemicals. In previous studies it has been shown that in neutrophils PCBs interfere with signaling involving phospholipase A2 (PLAZ) and calcium (Cali), two components Of the machinery required to fight pathogens. Accordingly, the overall goal Of the work outlined In this dissertation was to test the hypothesis that PCBs cause changes in Ca2+ homeostasis and phospholipase AZ by independent mechanisms and that these mechanisms can be triggered by any OC compound sharing a particular substructure similar to that found in ortho- chlorinated PCBs. TO provide the background and rationale for these studies, in the remainder Of this Introduction what is known about the physiology and mechanisms Of activation of neutrophils, with particular emphasis on PLAZ. will be discussed. In addition, a review Of the literature relevant tO PCBs, their chemistry and toxicity, and their effects on neutrophil function will be presented. Finally, the effects Of other OCs on cell activation will be reviewed. I.2. Neutrophils: Physiological function Neutrophils constitute the first line Of host defense against pathogens and are mediators Of antiviral immunity (Rouse et a/., 1978). Neutrophils are one Of the major cell types involved in inflammation. During tissue injury, locally released chemotactic factors recruit neutrophils from the bloodstream. Once at the site Of tissue damage, neutrophils undergo a series Of cellular changes leading to the killing and removal Of microorganisms. These processes are the result Of biochemical responses, and the most commonly Observed during neutrophil activation are superoxide anion production and degranulation. One Of the most studied neutrophil functions is degranulation. It is characterized by the release Of hydrolytic enzymes from membrane granules and intracellular vesicles and by the expression Of receptors and proteins on the plasma membrane (Fletcher et al., 1982; O’Shea et a/., 1985). The extracellular release Of specific granular constituents appears tO be crucial for the amplification Of the initial and subsequent phases Of the inflammatory response (Gallin et al, 1982). Degranulation can be triggered by a variety Of stimuli, such as chemotactic factors including formyI-methionyl-leucyl-phenylaIanine (fMLP) and C53 (Henson et al., 1978) Enzymes including lysozyme, B-glucuronidase and myeloperoxidase are released during degranulation. Neutrophils contain several types Of granules with particular compositions and release kinetics. Azurophilic granules contain myeloperoxidase, lysozyme and the membrane protein CD63 (Kuijpers et al., 1991). Myeloperoxidase generates hypochlorous acid and other chlorinated oxidants as both a host defense mechanisms and a means Of invoking tissue injury (Hazen et al., 1996; Weiss et al., 1983). In comparison to the azurophilic granules, the secretory vesicles are readily mobilized, and they contain cytochrome b558 and the heterodimeric glycoprotein CD11b/CD18 (Calafat et al., 1993). Tertiary granules store gelatinase, and their release upregulates CD11b/CD18 on the plasma membrane (Petrequin et al., 1987; Lacal et al., 1988). During microbial phagocytosis and exposure tO soluble stimuli, neutrophils undergo a rapid burst Of oxygen consumption leading to the generation Of oxidants. The main biochemical effector responsible for this respiratory burst is the superoxide-generating NADPH oxidase. This enzyme becomes activated when cytosolic and membrane bound proteins are assembled into a membrane complex that converts oxygen into superoxide anion. One Of the second messengers responsible for the activation Of NADPH oxidase is arachidonic acid (AA) (Henderson et al, 1993). The increase in NADPH oxidase activity elicited by AA is due to an increase in the number Of assembled enzyme complexes and to the augmentation Of its affinity for oxygen (Rubinek and Levy, 1993). I. 3. Neutrophils: Mechanisms of activation Neutrophil activation refers tO the series Of intracellular biochemical processes that follow exposure tO stimuli that are responsible for the detectable changes in neutrophil function, such as degranulation and superoxide anion production. The elucidation Of these processes is crucial to understand the mechanisms by which neutrophils participate in both host defense and inflammation. Mechanisms Of activation differ for different stimuli; however, similar biochemical pathways can be activated by chemically or physiologically unrelated stimuli (Lad et al., 1996). One Of the best documented compounds with respect to mechanism Of activation Of neutrophils is the chemotactic agent fMLP. FMLP activates neutrophils to undergo oxidative burst and degranulation. This process is mediated through a seven- transmembrane domain receptor coupled to a pertussis toxin-sensitive GTP binding protein, which in turn regulates Ca2+ mobilization and neutrophil function (Lad et al., 1985). The major components Of activation Of neutrophils by fMLP are Ca2+, phospholipase C (PLC), tyrosine kinase (TK), protein kinase C (PKC), phospholipase D (PLD) and phospholipase A2 (PLAZ). Each Of these will be discussed below in the context Of activation Of neutrophils by fMLP. It should be noted however, that these events are not unique to fMLP. Thus, this discussion can also be considered a framework for understanding intracelullar events during neutrophil activation by a variety Of stimuli, including some Of those examined in subsequent chapters. l. 3. A. Calcium FMLP stimulation Of human neutrophils leads to a rapid increase in the cytosolic free Ca” concentration ([Ca2+]i), an effect which is significantly reduced by removal Of extracellular Ca”. These results indicate that the increase in [Caz‘] is in part mediated by an increase Of the plasma membrane permeability to Ca”. The fMLP-dependent influx is not due to the activation Of voltage-dependent Ca2+ channels since depolarization did not affect the resting [Caz‘] (Andersson et al., 1986). Influx Of Ca2+ into fMLP-activated neutrophils is only detected on completion Of efflux Of the cation from intracellular stores (Anderson and Mahomed, 1997). The Observed influx is initially slow and is detected at 30-60 s after addition Of fMLP, accelerating around 2-3 min, and terminating at 5 min (Anderson and Mahomed, 1997). The long-sustained phase Of intracellular Ca2+ increase elicited by fMLP is reduced markedly by flunarizine, a blocker Of receptor-coupled Ca2+-channels. 1H-NMR studies have shown that flunarizine binds to cell membranes Of neutrophils and prevents Ca2+ entry (Pasini, et al., 1990). Similarly, adenosine causes a concentration-dependent inhibition Of the reactive oxygen species generated by fMLP-activated neutrophils and reduces the influx Of extracellular calcium induced by fMLP (Zhang et al., 1996). It is well established that human neutrophils have a least two distinct Ca2+ storage and release sites. One site is located peripherally next to the plasma membrane, and the other is in the juxtanuclear space. Confocal imaging has demonstrated that the non-peripheral Ca2+ storage site releases Ca2+ in response tO fMLP (Pettit and Hallet, 1998). In addition, phagocytosis relies mainly on Ca2+ release from internal stores which are replenished from the extracellular Ca2+ pool. The neutrophil has some ionomycin-sensitive Ca2+ stores which are not solicited during phagocytic activity. A gradient Of elevated [Ca2+], can be transiently Observed around the early phagosome, and changes in [Ca2+], are not a prerequisite for phagocytic activity (Theler et al., 1995). In addition to fMLP, other agonists can trigger changes in [Caz‘], Arachidonic acid derivatives such as hepoxilin A3 (HxA3), a 12— Iipoxygenase metabolite Of arachidonic acid, also can induce release Of Ca2+ in a concentration-dependent manner (DhO et al., 1990). Furthermore, platelet-derived metabolites such as 12-HETE (12- hydroxyeicosa(5Z,8Z,10E,14Z)tetraenoic acid) and 12-HPETE (12- hydroperoxyeicosa (5Z,82,10E,14Z) tetraenoic acid) can stimulate intracellular Ca2+ release in neutrophils, with 12-HETE being more potent than 12-HPETE. This is an example of transcellular modulation Of [Ca2+], in neutrophils (Reynaud and Pace-Asciak, 1997). Another metabolite Of AA, leukotriene B4 (LTB4) induces a rise Of [Car] in human neutrophils without involving pertussis toxin-sensitive G proteins or neutrophil activation (Palmblad et al., 1994). Finally, ryanodine, an agonist Of the ryanodine receptor, produces an increase Of [Ca2+]i due tO the liberation Of Ca2+ from internal stores and tO the influx Of extracellular Ca”. Neutrophils contain ryanodine—sensitive Ca2+ stores that might be involved in receptor- mediated chemotaxis (Elferink and De Koster, 1995). Interestingly, there is evidence suggesting that influenza A virus deactivates neutrophils via an increase in resting [CaZ+]i. It is suggested that high resting [Ca2+], has a negative effect on neutrophil function, specifically decreasing the ability tO kill bacteria (Haag-Weber and Horl, 1992) In short, after stimulation by agonist the biochemical behavior Of 2+]: diverse intracellular effector proteins requires changes in the [Ca and compartmentalization within the neutrophil. The source, magnitude and duration Of the Ca2+ oscillations ultimately determine the specificity in cellular function. I. 3. B. Phospholipase C Phospholipase C (PLC) activation results in hydrolysis Of phosphotidylinositOI-4,5-bisphosphate (PIPZ) to generate inositOl-1,4,5- trisphosphate (IP3) and 1,2-sn-diacylglycerol (DAG). In neutrophils, fMLP- stimulated PLC activation is mediated by a pertussis toxin-sensitive GTP- binding protein (Smith et al., 1985). IP3 can directly stimulate the IP3 receptor on the endoplasmic reticulum tO release Ca”. Preincubation Of neutrophils with an inhibitor Of PLCy, U73122, inhibits Ca2+ responses after neutrophil activation with fMLP, suggesting that PLC activation is necessary not only for Ca2+ release from intracellular stores but also for sustaining the extracellular influx of Ca” (Davies, et al., 1994). PLC signalling may be coupled to both PKC and PLD. DAG produced by PLC can activate PKC, and in turn PKC can enhance the response elicited by PLD (Exton, 1990). l. 3. C. Tyrosine kinases Activation Of tyrosine kinases in neutrophils mediates superoxide production (Tlthof et al., 1997), upregulation Of CD11b/CD18, adherence and locomotion (Naccache et al., 1994). It has been reported that stimulation Of neutrophils with fMLP leads tO a rapid and time- dependent increase in the tyrosine autophosphorylation activity Of the src— related tyrosine kinases lyn (Gaudry et al., 1995) and syk (Fernandez and Suchard, 1998), which may be involved in the activation and/or modulation Of enzymes/receptors located in close proximity such as NADPH oxidase and PLD. FMLP induces tyrosine phosphorylation and activation Of two distinct mitogen-activated protein kinases (MAPKs) with apparent molecular weights Of 40 kDa and 42 kDa, and treatment with genistein, a tyrosine kinase inhibitor, reduces phosphorylation Of the 40 and 42 kDa proteins (Torres 91‘ al, 1993). FMLP also activates p38 MAPK, which is an upstream activator Of NADPH oxidase (Lal et al., 1999). Genistein suppresses the InositOl phospholipid metabolism induced by the endoperoxide analog U-46619, suggesting that the turnover of inositol phospholipids is linked tO tyrosine phosphorylation (Gaudette and HOlub, 1990; Ozaki et al., 1993). Accordingly, activation of tyrosine kinases may lead tO release Of |P3 and subsequent rapid mobilization Of intracellular Ca2+ in neutrophils (Siddiqui and English, 1996). I. 3. D. Protein Kinase C PKC accounts for the majority Of the kinase activities Of neutrophils, and its intracellular distribution is to some extent dependent on 10 the state Of the activation of the cell. Following stimulation, the cytosolic PKC and a Ca2+-activated neutral proteinase (CANP) are translocated tO the plasma membrane where active CANP promotes proteolytic conversion Of PKC. Activated PKC is then released tO the cytosol, and it is fully active in the absence Of Ca” and phospholipids. PKC is involved in many steps Of Ca2+ signaling. For instance, different PKC isoforms require Ca2+ for their activity, and in turn PKC can phosphorylate Ca2+-ATPases (Becker, 1988). I. 3. E. Phospholipase D PLD catalyzes the hydrolysis Of phosphatidylcholine containing either ester- or ether-linkage at the sn-1 position into phosphatidic acid and DAG. PLD is involved in chemotaxis and superoxide anion generation induced by fMLP (Wanikiat et al., 1997). It has been shown that more than 90% Of the diglyceride formed in neutrophils in response tO fMLP is due tO activation Of PLD/phosphatidic acid phosphohydrolase (Billah et al., 1989a) which is sensitive tO a pertussis toxin-sensitive GTP-binding protein (Kanaho et al., 1991). In HL-60 granulocytes, the activation Of PLD requires both Ca” and GTP (Anthes et al, 1989) and can be both protein kinase-independent as well as protein kinase-dependent (Billah et al., 1989b). 11 l. 3. F. Summary Neutrophil activation by fMLP leads tO a rapid tyrosine phosphorylation followed by an increase in [Ca2+]i, activation Of phospholipases and protein phosphorylation, particularly stimulation Of PKC and the MAPK pathway (Figure I.1). These events precipitate release Of superoxide anion and degranulation. An additional pathway important in PMN stimulation is phospholipase A2 (PLA2) activation. Given the central role Of this enzyme in the studies described in subsequent chapters, PLA2 will be discussed in greater detail. I. 4. Phospholipase A2 and cellular function. PLA2 is the enzyme responsible for the release Of arachidonic acid (AA) from the sn-2-position Of phospholipids. Depending on the isoform, the substrate for PLA2 can be phosphatidylcholine, phosphatidylethanolamine, or plasmalogen, among others. In neutrophils, most Of the AA released during cellular stimulation is attributed tO the stimulation Of PLA2 (Chilton and Murphy, 1986). The activation Of PLA2 is one Of the primary steps in the production Of a broad spectrum Of inflammatory precursors in neutrophils. For instance, the release Of AA by PLAZ increases the activity Of the assembled NADPH oxidase in cytoplasmic membranes Of neutrophils to produce superoxide anion (Rubinek and Levy, 1993; Dana et al., 1998), an oxidant required for killing 12 flVILP Ca2+ Degranulation O .- ~-- 2 I. I . . «mama '41?- .M' «3‘ «va- 1» "As-lap. Iii-p‘-\|yth:vrll.n‘i:vfl- fip‘ f. III] “ultimatumImlilillinnjilil6i ,m ,..., :4. .m. w, tenet. .a. .,\:-l ._»- 3." mm“? m: ..I~e Deg-mu] alien Figure I.1. Signal transduction during neutrophil activation by fMLP pathogens. PLA2 activation has been linked to other cellular responses in stimulated neutrophils such as degranulation (Cockcroft, 1991) and adhesion and spreading (Chun and Jacobson, 1993). An excellent review about the regulatory functions Of phospholipase A2 was recently published (Murakami, et al., 1997). In general, PLAzs can be divided into extracellular (secreted) and intracellular isoforms. Secreted PLA2s (sPLAZ) are commonly called groups V and IIA secreted PLAzs. Intracellular PLAzs are present in the cytosol and include cPLAZ, referred tO as group IV and the Ca”- independent PLAZ (iPLAz) named group VI PLAZ. The different types Of phospholipases A2 have been reviewed (Bauldry et al., 1996; Bereziat, 1996; Murakami, et al., 1997, 1998). A summary Of their characteristics is presented in Table 1. 14 kD sPLAz is present in large quantities in human eosinophils (Blom et al., 1998). sPLA2 Is secreted during ischemia-reperfusion injury (SonninO et al., 1997). sPLAzs can differ significantly in their pH- dependence (Murakami et al., 1997b). The microtubular system Is necessary for the synthesis Of sPLAz induced by TNF-Ot along with IL-1[3 (Pruzanski et al., 1997), and after removal Of the cytokines the induced- sPLAz synthesis continues for over a period Of 8 hours (Vervoordeldonk er al., 1997). 14 Table |.1. Selected properties of Phospholipases A2 PLAz MW Tissue Ca2+ (kDa) Requirement References sPLAz 14 kDa Neutrophils Yes (mM) Rosenthal et al., 1995 14 kDa Pancreas Chang et al., 1999 16 kDa Colon Lamura et al., 1997 cPLA2 85 kDa Yes (nM) De Carvalho et al., 1995 CPLAZQ 85 kDa Yes Pickard et al., 1999 CPLA2B 114 kDa Yes Pickard et al., 1999 CPLAzy 61 kDa NO Pickard et al., 1999 iPLA2 85 kDa Pancreatic islets NO Ma et al., 1997, 1998 80 kDa Macrophage cell Ackermann et al., 1994; line P388D1 1995 85 kDa Myocardium Hazen et al., 1993 88 kDa Human Ma et al., 1999 B-Iymphocytes Activity Of cPLA2 can be modulated by protein phosphorylation involving the MAPK pathway (Qiu and Leslie, 1994). The cPLA2 sequence contains consensus phosphorylation sites for PKC, PKA, TK and MAPK (Sharp et al., 1991). cPLA2 can be phosphorylated in vitrO by the serine- threonine-specific kinases p42 MAPK and PKC (Nemenoff et al., 1993; Lin et al., 1993). In mammalian cells the common phosphorylation sites on cPLA2 are ser-505 and ser—727 (Borsch-Haubold, 1998). The activation Of cPLAz is mediated by an N-terminal domain which is required for the Ca2+- dependent translocation Of the enzyme to the membranes. The cPLA2 15 domain interacts primarily with the head group Of the phospholipid and prefers Ca2+ over other group IIA cations (Nalefski et al., 1998). This increase in [Ca2+], has tO be prolonged in order to translocate to cell membranes (Ishimoto et al., 1996). The binding motif Of cPLAz surrounds two adjacent CaZ+-binding sites together with an adjoining strip Of basic residues, which suggests that electrostatic and hydrophobic forces are important for membrane binding (Perisic et al., 1998). Interestingly, protein-protein interactions seem tO play a major role in regulation Of PLA2. For instance, in neutrophils, cPLA2 is directly inhibited by interaction with annexin V (Mira et al., 1997) and lipocortin l (Haigler et al., 1987). cPLA2 can also be regulated both transcriptionally and translationally, as seen for lL-1 B in rat mesanglial cells (Schalkwijk et al., 1993). cPLAz can be phosphorylated by a diverse number Of agonists. sPLA2 can trigger the phosphorylation Of cPLAz through a PKC/MAPK pathway (Huwiler et al., 1997). Macrophage cPLAz can be phosphorylated through a MAPK pathway by stimulation with phorbol 12-myristate 13- acetate (PMA), zymosan and LPS but not with the Ca2+-ionophore A23187 (Ambs et al., 1995), although there are MAPK-dependent and independent mechanisms Of activation Of cPLAz by LPS (Fouda et al., 1995). In platelets, collagen is able to stimulate the phosphorylation Of cPLA2 through both p38 and p42/p44 MAPK (Borsch et al., 1997). 16 In myocytes iPLAz exists as a 400-kDa complex made by a 40-kDa PLA2 catalytic subunit polypeptide and a tetrameric phosphofructokinase- related regulatory subunit (Hazen and Gross, 1993). In this system, iPLAz activity is strongly inhibited by calmodulin (Wolf and Gross, 1996). Despite the lack Of understanding Of the regulation Of iPLAz, a relatively selective pharmacological inhibitor exists. Hazen et al. (1991b) have found that BEL is >1000-fold more specific for inhibition Of iPLA2 than for the Ca2+- dependent PLAzs. This inhibition is both irreversible and time-dependent. Although BEL is selective for iPLA2 among PLAzs, BEL also inhibits cellular phosphatidic acid phosphohydrolase (PAP) activity in P388D1 macrophages. This inhibition results in the blockage Of triacylglycerol biosynthesis (Balsinde and Dennis, 1996). Annexins are proteins that participate in the modulation Of PLAzs. Annexin VI inhibits sPLAz by sequestering the phospholipid substrate (Koumanov et al., 1997). Epidermal and dermal PLA2 can be inhibited by annexin V, annexin II and annexin I by a mechanism which implies that there is no Interaction between PLAZ and annexins (Bastian et al., 1993). At least for annexin I, the inhibition Of PLA2 is dependent on the concentration Of Ca2+ in the media (Buckland and Wilton, 1998). Interestingly, annexin V uses the same mechanism to inhibit PKC (Dubois etaL,1998) 17 The differences in Ca2+ requirements, the biochemical specificity in terms Of regulation and substrates and the stability to certain agents such as disulfide-reducing chemicals has allowed the analytical differentiation Of all the intracellular and extracellular PLAzs (Yang et al., 1999). The work described in the following chapters tests the hypothesis that polychlorinated biphenyls alter cell signaling pathways dependent on Ca2+ and phospholipase A2 in rat neutrophils. Accordingly, background information on PCBs will be reviewed before presentation Of those studies. I. 5. Polychlorinated biphenyls I. 5. A. Structure Polychlorinated biphenyls (PCBs) are halogenated aromatic hydrocarbons with a biphenyl moiety in which hydrogens are substituted by chlorines (Figure l.2). Chlorine substitution in the biphenyl leads tO the formation Of 209 compounds named as congeners. Only about 130 individual congeners have been identified in commercial PCBs mixtures at concentrations 2 0.05% (Giesy and Kannan, 1998). I. 5. 8. Sources, distribution and occurrence PCBs were commercially produced beginning in 1929 and have been widely used industrially as dielectric fluids for transformers, plasticizers, cutting Oils, pesticide extenders, flame retardants, etc. 18 ( 6 , AE =14.7 Kcallmol \J V AE= 2.2 Kcallmol Figure |.1. Molecular structure of polychlorinated biphenyls. Computer modelling using the semiempirical method AM1 was used to fully Optimize the structure of PCB congeners. A. OrthO-chlorlnated 2,2’,4,4’-tetrachlorobiphenyl. B. Non-orthO-chlorinated 3,3’,4,4,- tetrachlorobiphenyl. Figure A shows the numbering position for the different atoms in the biphenyl, the torsional angle (a) generated between the two phenyl groups and the rotational energy barrier (AE) calculated as the difference between the energy Of the conformer in the planar versus the non-planar state. 19 (Hutzinger et al., 1974). In the environment PCBs are found as mixtures whose source is mainly commercial preparations (Tanabe et al., 1987). Commercial PCB mixtures known as Aroclors were formerly produced by the Monsanto Chemical Company in the US. (St Louis, MO), and in general they are named according to their percentage Of chlorine content by weight (Waid, 1987). For example, Aroclors 1221, 1242 and 1248 have 21, 42 and 48% chorine content by weight, respectively. PCBs exhibit a broad range Of physicochemical properties that account for their diverse biological and environmental distribution profile. PCBs have low water solubility, relatively low vapor pressure and extreme resistance to chemical reactions (Shiu and Mackay, 1986). These three properties are inversely related to chlorine content (Waid, 1987). Due to their lipophilicity and different rates Of metabolism, PCBs can be bioaccumulated and biomagnified in the food chain (Kannan et at, 1998; Matthews and Anderson, 1975), and this accounts for their persistence, widespread distribution, and mobilization to remote areas (Subramanian et al., 1983) such as the Himalayan lakes (Galassi et al., 1997) and Antarctica (Kallenborn et al., 1998). Humans can accumulate PCBs from direct exposure and by bioaccumulation processes through the diet (Patandin et al., 1999). PCBs have been found in breast milk (Ramos et al., 1997; Schecter et al., 1998), 20 blood, adipose tisssue, and placenta (Laden et al., 1999), among other fissues. 1. 5. C. Toxicokinetics Of PCBs In mammalian systems, PCBs can be metabolized via cytochrome P4503 1A1, 1A2, 2B1 and 282. These enzymes catalyze the formation Of intermediate arene oxides that are converted tO hydroxyl or methyl sulphone metabolites and subsequently into sulfate or glucuronide conjugates (Bergman et al., 1994). 1. 5. D. Toxicology Of PCBs The toxic effects Of PCBs have been discussed in different reviews (Safe, 1984; Silberhorn et al., 1990) and are widely diverse. PCBs can cause immunosuppression (Vos and Loveren, 1998), neurodevelopmental deficiencies (Jacobson and Jacobson, 1997), changes in sex hormone levels and sweet preference behavior (Hany et al., 1999a), hearing deficits (Goldey et al., 1995), periodontal diseases (Hashiguchi et al., 1999), dermatological problems (Nakayama et al., 1999) and neutrophilia (Hansen et al., 1995) among other effects. Although extensive epidemiological studies have revealed an association between PCBs and cancer (Cogliano, 1998), there is still an ongoing controversy about this effect (Moysich et al., 1999; Dorgan et al., 1999). 21 It has been shown that the presence Of congener mixtures in biological samples is similar tO that present in different commercial preparations (Dewailly et al., 1996). Different Aroclors have been reported to exert toxicological effects. Aroclor 1242 alters the levels Of thyroid hormones in rats (Morse et al, 1996a). Aroclor 1254 induces long-term changes in neuronal and glial cell proteins such as synaptophysin, glial fibrillary protein and calcineurin (Morse et al., 1996b) and alters serotonin metabolism (Morse et al., 1996c). Aroclors 1254, 1260, 1242 and 1016 induced hepatocellular neoplasms in animals (Mayes et al., 1998). Metabolism, toxic potential and mechanism Of action vary with physicochemical properties Of the congeners (Seegal, 1996; McFarland and Clarke, 1989). It has been proposed that the presence Of substitutions at particular sites on the phenyl ring determines the mechanism Of action for each congener. PCB congeners that are not orthO-substituted, such as those substituted in both para and two or more meta positions, appear to have greater affinity for binding to the Ah-receptor (AhR) and elicit effects similar tO those Observed for 2,3,7,8-tetrachlorOdibenzo-p-dioxin (TCDD), the compound with the highest affinity for the AhR (Kafafi et al., 1993a). These compounds are commonly referred to as dioxin-like PCBs. On the other hand, congeners with orthO-substitutions have low affinity for the AhR and consequently are called non-dioxin like PCBs. The differences in activity Observed with ortho and non-on‘hO- PCBs are in part due to 22 structural and electronic properties derived from the three-dimensional conformations generated by the presence Of chlorines in who positions. A chlorine in the orthO position Of a ring in the biphenyl skeleton interacts sterically with an hydrogen or another chlorine in the other ring Of the biphenyl creating a steric force that shifts one Of the rings out of the plane increasing the torsional angle between the two phenyl groups (Figure M). This is the reason why orthO- and non-orthO-PCBs are called coplanar and non-coplanar PCBs, respectively. Although this shifting occurs both in orthO- and non-orthO-PCBs, it is greater in on‘hO-PCBs because the force Of steric interaction between chlorines and chlorine/hydrogens is greater than for hydrogens alone. In fact, using computer modelling, it has been demonstrated that the majority of PCBs are likely to interact with the AhR in their non-planar conformation (Kafafi et al., 1993a) and that their interaction with the AhR depends on their lipophilicities, electron affinities, entropies and differences in energy Of frontier orbitals (Kafafi et al., 1993b). The structure Of representative ortho- and non-OIthO PCBs is shown in Figure 1. I.5.D.i. Toxicology Of dioxin-like PCBs Non-on‘ho chlorinated PCBs bind directly to the AhR and elicit a variety Of responses (Bandiera et al., 1982). These include induction Of hepatic drug metabolizing enzymes such as aryl hydrocarbon 23 hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (EROD) (Kafafi et al., 1993b; Sanderson et al., 1996), and arylamine oxidase (Marczylo and loannides, 1997), and alterations Of hepatic glucose 6-phosphate dehydrogenase (Hori et al., 1997). In environmental and human samples, several dioxin-like congeners are present, including 3,3’,4,4’- and 3,4,4’,5- tetrachlorobiphenyl, and 2,3,3’,4,4’- and 3,3’,4,4’,5-, pentachlorobiphenyl (Hong et al., 1993; Safe et al., 1985). 3,3’,4,4’-tetrachlorobiphenyl produces both an induction and inhibition Of cytochrome P4501A in a dose-dependent manner in vitrO (Hahn et al., 1993). This congener causes a decrease in the anti-sheep red blood cells antibody forming cell response and also produces thymic atrophy, hepatomegaly, hepatic centrilobular hypertrophy and extensive hepatic fatty infiltration (Silkworth and Grabstein, 1982). I. 5. D.ii. Toxicology Of non-dioxin like PCBs Although most on‘hO-substituted PCBs have low affinity for the AhR, they dO have biological activity. Cell stimulation by on‘hO- chlorinated PCBs involves the activation Of a series Of subcellular systems. Although a receptor-mediated mechanism has not been discovered, some studies have suggested that in fact orthO-PCBs can interact directly with cellular receptors (Angus and Contreras, 1995). A recent symposium 24 dealing with the mechanisms Of toxicity induced by of orthO-substituted PCBs has been published (Fischer et al., 1998). Effects Of on‘hO-substituted PCBs have been documented in cells from the central nervous system (Kodavanti et al., 1997). Structure-activity relationships have shown that congeners with one, two or three chlorine substitutions and having a log Kow between 5.2 and 6.6 are more active than other congeners (Svendsgaard et al., 1997). Aroclor 1016 and ortho- substituted PCB congeners decrease dopamine concentration in the caudate, putamen, substantia nigra, and hypothalamus (Seegal et al., 1990). Aroclor 1254 has the same effect in PC12 cells (Angus and Contreras, 1994). PCB mixtures Aroclors 1016, 1254 and 1260 and individual congeners with on‘hO/meta- or on‘hO/para- chlorine substitutions inhibit cerebellum microsomal and mitochondrial Ca2+-sequestration (Kodavanti et al., 1996). In cerebellar granule cells, 2,2’-dichlorobiphenyl causes a 2+]i, an inhibition Of microsomal concentration-dependent increase in [Ca and mitochondrial Ca2+-sequestration (Kodavanti et al., 1993; Shafer et al., 1996), a biphasic effect on receptor-mediated phosphatidylinositol hydrolysis and translocation Of PKC (Kodavanti et al., 1994). PCBs containing two or more orthO-chlorine substituents activate ryanodine receptors (RyRs) in mammalian brain, mobilizing Ca2+ in a dose- dependent manner, whereas the non-orthO-substituted PCBs are 25 completely inactive (Wong et al., 1997a). The mechanism responsible for this effect has been proposed to involve the disruption Of the modulating effect of immunophilin FKBP12 on the distinct conformations Of the Ry1R complexes (Wong and Pessah, 1997). At the transcriptional level, the orthochlorinated 2,3,4,2’,4’,5’ hexachlorobiphenyl (PCB-138) induced overexpression of res, jun, and myc in the 3T3-L1 cell line (Gribaldo et al., 1998). 2,3’,4,4’,5- pentachlorobiphenyl induced methylcholanthrene- and phenobarbital- inducible isoenzymes Of cytochrome P450 monooxygenases and enhanced foci growth in rat liver (Haag-Gronlund et al., 1997). OrthO-chlorinated PCBs can abrogate some Of the toxic responses Observed with non-orthO-chlorinated PCBs. For instance, 2,2’,4,4’,5,5’- hexachlorobiphenyl protects from 3,3’,4,4’,5-pentachlorobiphenyl-induced embryo malformations (Zhao et al., 1997). Other non-dioxin-Iike responses Of PCBs include estrogenic activity both in vivo and in vitrO (Arcaro et al., 1999). Interestingly, hydroxylated, non-orthO and OIthO-chlorinated PCBs have been reported tO have antiestrogenic activity in vitrO (Kramer et al., 1997) and in vivo (Connor et al., 1997). In the immune system, the effects Of PCB have been extensively studied in neutrophils. The next section will present a review Of what is known about the effects Of orthO-chlorinated PCBs in neutrophils. 26 1.5.E. OrthO-chlorinated PCBs and neutrophil function On‘hO-chlorinated PCBs cause superoxide anion production, degranulation and inhibition Of fMLP-induced degranulation in rat neutrophils (Ganey et al., 1993). These processes are mediated through Ca2+-dependent mechanisms (Brown and Ganey, 1995) and are elicited only by OIthO-chlorinated biphenyls (OliverO and Ganey, 1998; Brown et al., 1998). The PCB mixture Aroclor 1242 induces an increase in intracellular calcium in rat neutrophils (See Chapter II). The time course Of the increase in [Ca2+], shows a delay and starts only after 5 minutes incubation with Aroclor 1242, reaching a maximum after approximately 15 minutes. Furthermore, Aroclor 1242 or 2,2’,4,4’-tetrachlorObiphenyl but not 3,3’,4,4’-tetrachlorObiphenyl induces the production Of inositol phosphates in neutrophils, suggesting activation Of PLC (Tlthof et al., 1995). PCBs induce the activation Of Ca2+-independent PLAZ in neutrophils. This effect has been Observed for on‘hO-substituted PCBs but has not been detected for non-orthO-substituted PCBs (Tlthof et al., 1998). This Aroclor 1242-induced PLA2 activation is linked tO generation Of superoxide anion from rat neutrophils, and inhibition Of PLA2 activity with BEL also inhibits superoxide anion production (Tlthof et al, 1996). Conversely, TCDD, which binds specifically to neutrophils from Ah-responsive mice, does not impair the production Of superoxide anion and hydrogen peroxide or 27 degranulation, measured as the release Of B-glucoronidase (Ackerman et al., 1989). The production Of superoxide anion in neutrophils exposed to on‘hO-PCBs has been linked to activation Of PLC (Tlthof et al., 1995), TK (Tlthof et al., 1997; VOie et al., 1998) and PLD (VOie et al., 1998). 1. 6. Cell activation by other chlorinated chemicals Other chlorinated chemicals affect signal transduction pathways similar to those activated in neutrophils by PCBs. Hexachlorocyclohexanes (HCCHs) are a group Of chemicals widely used as pesticides. Theoretically there are eight possible conformational isomers for these compounds, the most common being or, [3, 5 and y. The y-isomer is commonly known as Iindane and has been shown to induce hepatocellular carcinoma and lung cancer (Wolff et al., 1993). In humans, symptoms Of Intoxication with Iindane include seizures Of the mixed type, intention tremors, memory impairment, and irritability. The a-, y-, and 8-, but not B-HCCH, induce superoxide anion production and release Of intracellular Ca2+ in human neutrophils (Kuhns et al., 1986). y-HCCH-induced superoxide production is sensitive to the intracellular antagonist Of Ca2+ release, TMB-8, suggesting a role for intracellular Ca2+ in superoxide production by y—HCCH. Lindane induces superoxide anion release by a mechanism that does not involve a physical 28 interaction Of the agonist with the NADPH-dependent superoxide- generating system (English et al., 1986). It has been reported that y-, and 8-HCCH isomers, which stimulate superoxide formation, inhibit chemotaxis to other mediators as a result Of widespread and dysfunctional changes in intracellular calcium that can no longer effect the coordinated cytoskeletal actions required for cell movement (Kaplan et al. 1988). Structurally, HCCHs resemble analogs Of inositol. For instance, muco— and myO-inositol have the stereochemical configuration Of y and 5 isomers Of HCCH, respectively. Mohr et al. (1995) demonstrated that the 8- isomer Of HCCH releases Ca2+ from IP3-sensitive Ca2+ stores, and that this is not due to its similarity to myO-inositOI-1,4,5-trisphosphate. 6-HCCH did not compete with IP3 for the IP3 receptor, suggesting the presence Of a binding site for 5-HCCH in IP;, sensitive Ca2+ channels. Lindane induces profound effects on the phosphatidylinositol cycle through the generation Of phosphatidic acid and consumption Of PIPZ (English at al., 1986) and decreases the incorporation Of myO-[2-3H]inositol into phosphoinositides (PI) with some degree Of specificity depending on the different Pl classes (CarrerO et al., 1996). In myocytes, the disruption Of PI-generated second messengers by Iindane can lead to inhibition Of gap junctional communication (Criswell et al., 1995), as Observed in other cell types (Guan and Ruch, 1996). Because EDTA prevented the effects 29 Of Iindane on PI synthesis, it was hypothesized that Iindane inhibits phosphatidylinositol synthesis by increasing intracellular calcium. This increase in intracellular calcium may be due to two factors. First, in macrophages, Iindane can directly trigger a Ca2+ influx (Pinelli et al., 1994). Second, this Ca2+ can in turn activate PLC to generate a sustained increase in cytoplasmic free Ca2+ after IP3 generation. Furthermore, EGTA blocks the Iindane-induced [Ca2+]t increase, suggesting a role for extracellular Ca2+ during neutrophil activation by this compound (Grigorian et al., 1988). The increase in [Ca2+]i together with the inhibition Of Ca2+,Ki- ATPases may account for the toxic effects Of Iindane in the cardiovascular system Of rats (Anand, et al., 1995). At concentrations greater than 50 uM, Iindane activates a phospholipase A2 (PLA2) which prefers phosphatidyl inositol rather than phosphatidyl choline as substrate (Lopez-Aparicio et al., 1995). PLA2 activity in both soluble and membrane fractions was not modified by Iindane (30-300 uM) over a 120 min period, suggesting a role for particular intracellular messengers in PLA2 activation. On the other hand, Iindane can directly stimulate PKC activity (Bagchi, et al., 1997). Interestingly, Enan and Matsumura (1998) have shown that c-neu tyrosine kinase is activated by B-HCCH in cell-free and intact cell preparations from MCF-7 human breast cancer cells. The effects Of dieldrin on neutrophil function are not as well documented as those for Iindane. Dieldrin causes a concentration-dependent increase in superoxide anion production by 30 neutrophils, which is regulated by extracellular calcium (Hewett and Roth, 1988). It has been suggested that dieldrin activates PLC (De Schroeder and De D’angelo, 1995) and inhibits forskOlin-induced stimulation Of adenyl cyclase (Carrero et al., 1993). I. 7. Summary In summary, PCBs stimulate neutrophils and in doing so invoke a number Of signal transduction pathways involved in activation Of the cells by other agents. These include PLC, TKs, PLA2 and increased intracellular calcium. Similar pathways may be important in HCCH- and dieldrin- induced stimulation Of neutrophils. Activation Of PLA2 is critical and appears to be an early event after exposure tO PCBs. The PLA2 activated is Ca2+-independent, and regulation Of iPLAZ is not understood. Accordingly, studies have been undertaken to elucidate the mechanisms Of activation Of iPLA2 during neutrophil activation by PCBs and other organochlorine compounds. These studies are described in detail in the following chapters. The overall hypothesis tested by these studies is that PCBs cause changes in Ca2+ homeostasis and phospholipase A2 by independent mechanisms and that these mechanisms can be triggered by any OC compound sharing a particular substructure similar to that found in OlthO-chlorinated PCBs. 31 Chapter II REGULATION OF CALCIUM HOMEOSTASIS BY POLYCHLORINATED BIPHENYLS IN RAT NEUTROPHILS: EFFECTS ON REPONSES TO THE CHEMOTACTIC AGENT FMLP 32 ".1 Summary Polychlorinated biphenyls (PCBs) activate polymorphonuclear neutrophils (PMNs) acting on different subcellular targets. The Objective Of this study was tO characterize the effects Of PCBs on Ca2+ homeostasis in rat PMNs. Aroclor 1242, a technical PCB mixture, induced a dose- and time-dependent increase in the concentration Of intracellular free calcium ([ca2+ 1,) in Fura 2/AM-labeled PMNs. A significant increase in [Ca2+], from basal levels was Observed only after 5 min and reached a plateau after 10- 15 min. TO examine whether the effects Of PCBs on Ca2+ homeostasis are structure-dependent, studies were performed with individual congeners. 2,4’-dichorobiphenyl, an OIthO-PCB, produced the same effect on Ca2+ as Aroclor 1242, whereas 3,3’,4,4’-tetrachlorObiphenyI, a non-ortho PCB congener, was inactive. N-formyl-methionyI-leucyl-phenylalanine (fMLP) induced a biphasic increase in intracellular Ca”. The first phase is due tO release Of Ca2+ from intracellular stores, while the second one is due to extracellular Ca2+ influx. The rapid increase in intracellular Ca2+ elicited by fMLP during the first phase was abrogated in a time-dependent manner by Aroclor 1242, suggesting that the intracellular calcium stores activated by both Aroclor 1242 and fMLP are the same. Furthermore, Aroclor 1242 blocked Ca2+ influx induced by fMLP. In short, the PCB-induced increase in [C321, in PMNs was dose-, time- and structure-dependent, and the Ca2+ pOOl released was the same store as that released by fMLP. 33 ".2. Introduction Among environmental pollutants, PCBs are Of great concern due to their global distribution, high persistence, and the toxicological diversity in terms Of congener specificity. PCBs can be categorized in two different groups depending on the presence or absence Of a chlorine atom in the 2- position Of either Of the phenyl groups composing the biphenyl. In general, it is accepted that PCBs lacking the chlorine atom in the 2-position can Obtain a quasi-planar conformation allowing for their interaction with the Ah receptor (Ah-R) (McKinney and Waller, 1994). On the other hand, those PCBs with a chlorine group in the 2-position (orthO-chlorinated PCBs) are commonly referred to as non-coplanar and do not exhibit high affinity for the AhR. Although the orthO-chlorinated PCBs have low potency in inducing AhR-mediated effects, their toxicological profile is diverse and includes neurotoxicity (Kodavanti et al., 1994; Choksi, et al., 1997; Tilson and Kodavanti, 1997), cancer promotion (Wolfle, 1997-98), inhibition Of cell-cell communication (Kato et al., 1998) and neutrophil dysfunction (Ganey et al, 1993). One Of the Ah-R-independent biochemical effects Of PCBs is the alteration Of calcium (Ca2+) homeostasis in various cell types. PCBs cause changes in microsomal Ca2+ transport by direct interaction with ryanodine receptors in mammalian brain (Wong et al., 1997b), inhibition Of Ca2+ sequestration in rat cerebellum (Kodavanti, et al., 1996) and Ca”- 34 dependent induction Of insulin release from RINm5F cells (Fischer et al, 1996). In neutrophils, activation by PCBs is a Ca2+-dependent process that also involves other subcellular systems such as tyrosine kinases (Tlthof et al., 1997), phospholipase A2 (Tithof et al., 1996) and phospholipase C (PLC) (TithOf, et al., 1995). This recognized role Of Ca” in cell homeostasis can be attributed to its interaction with many proteins tO guarantee optimal cell function. Proteins that directly interact with Ca2+ include calmodulin, annexins, the EF-hand calcium-binding protein S-100, the Ca2+ binding C-2 domains and other extracellular proteins such as blood clotting and immune system proteins (Willians et al., 1998). The Objectives Of this work were tO examine the effects Of the commercial PCB mixture, Aroclor 1242, on Ca2+ homeostasis in neutrophils and to determine the effects Of these changes on the response elicited by the bacterial chemotactic compound fMLP. Results Of these studies will Offer clues about the role of alterations in Ca2+ homeostasis in neutrophil dysfunction induced by PCBs. II. 3. Materials and Methods II.3.A. Chemicals PCB congeners 8 (2,4’-dichlorobiphenyl) and 77 (3,3’,4,4’- tetrachlorobiphenyl), (>99% pure) and the PCB mixture Aroclor 1242 were purchased from Chemservice (West Chester, PA). Glycogen (Type 35 II from oysters) and fMLP were purchased from Sigma Chemical CO. (St. Louis, MO). II.3.B. Isolation Of rat, peritoneal neutrophils After glycogen elicitation into the peritoneum Of male, Sprague-Dawley, retired breeder rats, neutrophil isolation was conducted as described (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.35, containing 1.6 mM CaClz. The percentage Of neutrophils in the cell preparations was > 95%, and the viability was >95% determined by the ability to exclude trypan blue. Concentration Of cells in all Of the assays was 2 x 106 cells/mL. The isolation procedure was performed at room temperature. II.3.C. Measurement Of [Ca2+]. in rat neutrophils Neutrophils (2.5 x 106 cells/mL) were labeled by incubation for 25 min at 37 °C with 5 pM Fura-2/AM in HBSS. After loading cells were washed with HBSS, and the cell count was readjusted tO 2 x 106 cells/mL. Fluorescence emission at 505 nm was monitored at room temperature with constant stirring, using a dual wavelength spectrofluorometer system with excitation at 340 and 380 nm. The [Ca2+]i was calculated from fluorescence intensity readings using the following equation: [Ca2+]=Kd*Q(R-Rm,n)/(Rmax- R). R is the ratio Of emission intensities at 340 and 380 nm excitation 36 (340/380), Rm,“ is the 340/380 ratio under Ca2*-free conditions, Rmax is the ratio under saturating Ca2+ concentrations; Kd is the dissociation constant Of the Ca2+: Fura-2 complex; and Q is the ratio of the 380 nm fluorescence under conditions Of minimum and maximum [Ca2+]i conditions (Shao et al., 1998). The equilibrium dissociation constant, Kd, was taken from literature, 224 nM (Kankaanranta et al., 1995a). Rmax and Rm.n values for each assay were determined from the fluorescence intensities in the presence Of 0.01% Triton X-100 or 10 mM EGTA, respectively. These two parameters did not change significantly in the presence Of Aroclor 1242. Fluorescence emission after excitation Of 360 nm was monitored and in all studies conducted remained constant during data collection. Increases in resting [Ca2+]i evoked by agonists were detected by measuring the change in fluorescence ratio after exposure with the vehicle N,N-dimethyllformamide (DMF). Typical values for quiescent neutrophils ranged from 50 tO 100 nM. PCBs were added to labeled cells 50 sec after the start Of data collection. PCB stock solutions were prepared by dissolution Of the neat chemical in DMF, and 1 uL/mL Of the respective stock solutions was added to the cells to achieve the desired concentration in a final cell volume Of 3 mL. When inhibitors were used, labeled cells were pre-incubated with the inhibitor for 20 minutes at 37 °C before PCBs were added. 37 II.3.D. Statistical analysis Data are presented as the means i standard error Of the mean (SEM) for at least four different replicates. Comparisons between the calcium concentrations for different agonists at a particular time were done using the Student’s t-test. The criterion for statistical significance was p<0.05. ".4. Results lI.4.A. Aroclor1242-stimulated increases in [Ca2“]i in rat neutrophils are both time- and dose-dependent Aroclor 1242 and the PCB congeners 2,4’-dichlorobiphenyl (PCB 8) and 3,3’,4,4’-tetrachlorobiphenyl (PCB 77) were investigated to determine their capacity to induce changes in [Ca2+]i in rat neutrophils. Aroclor 1242 induced a time-dependent increase in [Ca2+], in rat neutrophils. Significant changes from basal levels were Observed after 5-6 minutes incubation, and [03”], continued to increase through 15 minutes (Figure Il.1). This increase in [C3215 was also dose-dependent (Table Il.1). Addition Of EGTA 50 seconds before treatment with agonists abolished the second phase Of fMLP-induced increase in [Ca2+], but did not eliminate the Aroclor 1242-induced response (Figure "2), although the net increase was reduced (Figure ”.2, compare to Figure Il.1). These results 38 400- E 300 - 5 Aroclor 1242 jf='2oo- N CB 2. 100 - y DMF I r I I I I I o 150 300 450 600 750 900 Time (sec) Figure ".1. Effect of 10 pg/mL Aroclor 1242 on [Ca2+], in rat neutrophils. Cells were loaded with fura-2/AM as described in Materials and Methods. Fura-2-Ioaded neutrophils were stimulated with 10 ug/mL Aroclor 1242 at the time indicated by the arrow. Data for control (DMF) are shown for comparison. Results are expressed as means i SEM for four different experiments. 39 Agonist Incubation time (minutes) Concentration 5 10 15 Aroclor 1242 1 pg/mL 4.4184 29.0:102 59.1:t13.7 5 pg/mL 20.6:54 60.6i7.1 83.0_+_12.7 10 ug/mL 28.3:16 125.5:85 199.4:44 FMLP 2 pM 284.9i49.5 287.4:388 170.1i12.6 Table ".1. Net changes in basal [Caz‘]; (nM) in rat neutrophils exposed to different concentrations of Aroclor 1242. Data are presented as the mean i standard error for at least three different experiments conducted as presented in Materials and methods. FMLP data are presented for comparison. The maximal change in increase in [Ca2+], induced by fMLP was Observed at 7 min treatment (351.2:718 nM). 40 nM) V H + [Ca2 200 . —0— Aroclor1242 —0— fMLP 180 160 140 120 100 80 -w-— DMF 0 150 300 450 600 750 900 Time (sec) Figure ".2. Effect of EGTA on Aroclor 1242- and fMLP-induced increase on [Ca2+],- in rat neutrophils. Cells were labeled with fura- 2/AM as described in Materials and methods. Fura-2-Ioaded neutrophils were treated with 5 mM EGTA and 50 seconds later with either 2 uM fMLP, 10 ug/mL Aroclor 1242 or 1 pL/mL DMF (vehicle control). Results are expressed as mean 1r SEM for four different experiments. 41 show that Aroclor1242-induced increase in [CaZ+]i is both dose- and time-dependent and Is decreased in magnitude, but not abolished, in the absence Of extracellular Ca”. "43. OrthO-chlorinated but not non-orthO-chlorinated PCBs reproduce the effects of Aroclor 1242 The on‘hO—chlorinated PCB 2,4’-dichlorobiphenyl caused an increase in [Caz‘] similar tO that Observed with Aroclor 1242; however, the non-orthO—chlorinated PCB 3,3’,4,4’-TCB, failed tO induce a change in [Ca2+], (Figure ”3). These results are in agreement with previously reported data showing that in human neutrophils orthO-substituted PCBs but not non-orthO-PCBs increase [Ca2+]i (VOie et al., 1998). I|.4.C. Aroclor 1242 interferes with fMLP-induced increases in [Ca2+]i Treatment Of neutrophils with the chemotactic agent fMLP causes a characteristic, time-dependent, biphasic increase in [Ca2+]i. Data from our lab represent this well (Figure ”4). The first phase, which results from a rapid release Of intracellular Ca2+, lasts for approximately 50-80 seconds. The second phase, which starts at about 2-3 min (Figure “4), is characterized by a bell-shaped increase in [0321i which lasts for about 13 minutes, reaching a maximum at 7. It is well recognized that the first phase 42 ] (HM) [Ca2+ 300 - 250 . - 3,3',4,4'-tetrachIorobiphenyl - 2,4'-dichlorobiphenyl 200 ‘ 150 ' 100 " 50 I n"! 0 . 0 150 300 450 600 750 900 Time (sec) Figure ".3. Effects of 2,4’-dichlorobiphenyl and 3,3’,4,4’- tetrachlorobiphenyl on [Ca2+], in rat neutrophils. Cells were labeled with fura-2/AM as described in Materials and Methods. Fura- 2-Ioaded neutrophils were stimulated with 25 uM Of the indicated congener at the time indicated by the arrow. Results are expressed as means i SEM for four different experiments. 43 [Ca2+], (nM) + fMLP + Aroclor1242 + fMLP 500 - -l- Aroclor1242 400 - fMLP+ 300 -Aroclor1242 200 - 100 l 0 . 0 150 300 450 600 750 900 Time (sec) Figure ".4. Effects of simultaneous addition of fMLP and Aroclor 1242 on [Ca2+ ,- in rat neutrophils. Cells were loaded with fura-2/AM as described in Materials and Methods. Fura-2-loaded cells were stimulated simultaneously with 2 uM fMLP and 10 ug/mL Aroclor 1242. Data for 2 uM fMLP and 10 pg/mL Aroclor 1242 are shown for comparison. Results are expressed as means 1 SEM for four different experiments. 44 is due to the release Of Ca2+ from an inositol trisphosphate (IP3)-sensitive Ca2+ pool and the second phase is the result Of Ca2+ influx from extracellular Ca2+ upon discharge Of the intracellular stores (Anderson and Mahomed, 1997; Montero et al., 1991; Krause et al., 1993). These reports are supported by our Observation that EGTA abolished the second phase Of the fMLP-stimulated increase in [Ca2+]i but did not affect the first phase (Figure ”2). TO determine whether changes in [Ca2+], induced by Aroclor 1242 would affect the increase in [CaZ+]i stimulated by fMLP, experiments were performed in which cells were initially exposed tO Aroclor 1242 and then treated with fMLP or vice-verse. In the absence Of Aroclor 1242, [Ca2+], increased in a biphasic manner after addition Of fMLP as described (Figure ”4). Simultaneous addition of Aroclor 1242 and fMLP induced an increase in [Ca2+], during the first phase that was similar to, but less pronounced than, that Observed with fMLP alone and a significantly decreased rise in the second phase (Figure "4). Addition Of fMLP two minutes after exposure to Aroclor 1242 induced an immediate increase In [Ca2+], which emulated the first phase seen with fMLP alone. This was followed by a very small, second increase (Figure ll.5.A). A similar but less pronounced effect was Observed when fMLP was added five minutes after Aroclor 1242 (Figure “5.8). However, when fMLP was added after 45 .Amodvav OE: New? .2003 @2688. SO: Emumtzu $58555 .s .comtdeOO 6.. cameo some CO 8839 2m mcofi meme 6603 5:5 mzsmom .mEoEtoaxo Eoemtfi 62:38 LO“— Emm h wcmoE mm. commoaxo 2m mzzmom .wsotm 65 >9 cofiofiE mm n35: .21 N 53> Loam. woSEE g cm“ .0 av cé As 92 can News .282 uses 2 5s, accessed 2c; 2.8 Encodes”. .8262 cam circa—2 E 89.8% mm SENSE 53> nonmo. mum; m=oo .macqohaoc “E 5 Sang :O «32. L283. Ow 2:886 Leta 665.: “tweets we boaqqm n33: go wootm .m... 2:9“. O can. can Aces as; sue me: Be ems es em... 8» e... e e8 e2 8e 84 e8 a: e 8e 8e 8e 8.. 8e 8e e a. k a. as as Near 6.002. New? «3.. A, 3.093 8.25 Es: 52. £45: 0 m < atmwfin. .8—. sees (WU) leeO] 46 ten minutes Of exposure to Aroclor 1242 (Figure ll.5.C) no significant early increase in [Ca2+]i was detected. Reversing the order Of agonist incubation showed that when Aroclor 1242 was applied exactly at the end Of the first phase elicited by fMLP, most Of the second phase Of the response (Ca2+ influx) was blocked (Figure ”6). This Observation suggests that Aroclor 1242 is interfering with the calcium channel functioning on the cell membrane. ".5. Discussion In neutrophils, increases in [Ca2+]i have been Observed not only with the bacterial chemotactic agent fMLP but also with other pathogen-related agonists including influenza virus (Hartshorn et al., 1995), influenza A nucleoprotein (Cooper et al., 1996), virus T21/DP107, and a synthetic Ieucine zipper-like domain Of the HIV-1 envelope gp41 (Su et al., 1999). Results presented here demonstrate that PCBs increase [Ca2+], in rat neutrophils in a time-, concentration-, and structure-dependent way. Because Aroclor 1242-induced increases in [Ca2+]. were significantly different from basal levels only after approximately 4-5 minutes and increased progressively up to 15 minutes, it is reasonable to speculate that this event may be associated with the generation Of superoxide anion, which is significant after 10 min Of cell treatment with Aroclor 1242 (Ganey eta/.,1993). This interpretation is consistent with the Observation that 47 + lep + Aroclor1242 (2 min) 500 - + lep . A l 400 :22? E 300 " lep \l/ 5 £4" 200 - 3.; m . 2, 100 - V 0 . 0 200 400 600 800 1000 Time (sec) Figure ".6. Effect Of Aroclor 1242 applied two minutes after exposure to fMLP on [Ca2+],- in rat neutrophils. Cells were loaded with fura-2/AM as described in Materials and Methods. Fura-2- loaded cells were stimulated with 2 pM fMLP and two minutes later with 10 ug/mL Aroclor 1242 as indicated by the arrows. Results are expressed as means i SEM for five different experiments. 48 PCB-stimulated superoxide anion generation is Cay-dependent (Brown and Ganey, 1995). One Of the best characterized mechanisms Of increasing [Ca2+], by an agonist is that elicited by fMLP, which releases Ca2+ from intracellular stores. It has been well documented that neutrophils have at least two distinct Ca2+ storage and release sites. One site is located peripherally under the plasma membrane, and the other is in the juxtanuclear space. Confocal imaging has demonstrated that the central Ca2+ storage site releases Ca2+ in response to fMLP (Pettit and Hallet, 1998) after PLCy activation and release Of IP3 (Davies et al., 1994). The Ca2+ store released by fMLP is deep within the neutrophil, and the cloud Of elevated Ca2+ released from the store does not significantly affect the concentration Of Ca2+ near the membrane (Davies and Hallett, 1996). During the first 5 min Of exposure tO Aroclor 1242, co-treatment with fMLP triggers an increase in intracellular calcium that is significantly different from that Observed with Aroclor 1242 alone. However, when the release Of [Ca2+]; by Aroclor 1242 has reached a plateau (10-15 minutes), addition Of fMLP does not produce a further increase in [Ca2+],. One interpretation for this Observation is that the Aroclor 1242-sensitive Ca2+ store is the same that is targeted by fMLP. As a result Of a progressive depletion Of these stores by Aroclor 1242, the effect Of fMLP in producing a direct release Of intracellular Ca2+ from the same store is abrogated in a 49 time-dependent manner. That the source Of Ca2+ released upon Aroclor 1242 stimulation is intracellular is also suggested by the Observation that [Ca2+]; does not increase when Aroclor 1242 is added. to fMLP-treated cells at a time when fMLP has caused intracellular stores to empty and before refilling Of these from extracellular sources of Ca2+ has begun (Figure ”6). Further support arises from the lack Of effectiveness Of EGTA to block completely the Aroclor 1242-induced response. It is important, however, to point out that the use Of EGTA data to rule out the intra- or extracellular- dependent source Of Ca2+ in cell signaling should be interpreted with care. Neutrophils transferred tO CaZ+-free medium progressively lose their responsiveness to fMLP (Gabler et al., 1986), suggesting that there exists a rapid, passive mobilization from intracellular stores tO the extracellular space when a concentration gradient is present. An alternative explanation for the lack Of effect Of fMLP after 10 mins incubation with Aroclor 1242 is that increases in [Ca2+]i coming from a different store or extracellularly might regulate the IP3 receptor to inhibit further stimulation by IP3. The presence Of Ca2+ binding sites in the IP3 receptor (Patel et al., 1999) permit this possibility. However, this is unlikely because Aroclor 1242 failed tO increase [Ca2+]i further after depletion Of intracellular Ca2+ stores by fMLP (Figure ”6). As mentioned before, fMLP releases Ca2+ from an IP3-sensltive intracellular Ca2+ store. Although the results discussed above seem in 50 agreement with the interpretation that Aroclor 1242 shares the same, central Ca2+ pool as fMLP, the time course associated to each chemical suggests different mechanisms are involved in release from the pools by these two stimuli. Despite this, both fMLP and Aroclor 1242 induce a very early (15-20 sec) and similar release Of IP3 (TithOf, et al., 1995). It can be speculated that the absence Of an early rise in [Ca2+]i in Aroclor 1242- treated neutrophils in the face Of early release Of IP3 is due to a change in the IP3 receptor (IP3R) functioning mediated by Aroclor 1242. However, the increases Of [Ca2+]i induced by fMLP during the first 5 minutes Of treatment with Aroclor 1242 suggests that the IP3R is functional, and consequently Aroclor 1242 may not be interfering with the interaction Of IP3 and its receptor. An alternative hypothesis could be compartmentalization for different PLCs stimulated by Aroclor 1242 and by fMLP. Accordingly, although an early release Of IP3 will be Observed with both agonists, the IP3 released by Aroclor 1242 will not diffuse rapidly into the cytosol and will require accumulation over time tO reach the IP3R. On the other hand, the IP3 released by fMLP could be localized in the same compartment as the IP3R and elicit a rapid release Of Ca2+. Another possibility is that Aroclor 1242 acts directly on ryanodine receptors (RyRs), which have been identified in the neutrophil (Rardon and Krause, 1992; Elferink and De Koster, 1995), tO release Ca2+. This has been Observed in other tissues 51 such as the hippocampus and skeletal and cardiac muscles (Wong and Pessah, 1996; Wong etal., 1997a; Wong eta/., 1997b). Aroclor 1242 blocked most Of the Ca2+ influx characteristic Of the second phase Of the fMLP response. Because this phase arises from influx Of extracellular Ca”, this Observation suggests that Aroclor 1242 is interfering with the Ca2+ channel functioning on the cell membrane. This Observation may shed light on the mechanisms by which Aroclor 1242 and orthO-chlorinated PCBs block fMLP-induced degranulation as reported previously (Ganey et al., 1993; Olivero and Ganey, 1997). Although fMLP-induced degranulation is not totally abrogated by chelators Of extracellular Ca”, the granule release process is optimal when extracellular Ca2+ is present. The inhibition Of the fMLP-induced Ca2+ influx and the consequent decrease in neutrophil degranulation has been Observed for other compounds as well, such as adenosine (Pasini et al., 1990; Thiel and Bardenheuer, 1992), fenamates (Kankaanranta, et al., 1995a-b), trifluoperazine and W-7 (Capuozzo et al., 1997; Smith et al., 1981; Blackwood et al., 1995). Increases in [Ca2+], are considered to be essential for activation (Korchak et al., 1984) and subsequent bacteria killing by neutrophils (Wilsson et al., 1996); however, these events by themselves are not sufficient for the optimal neutrophil capacity for degranulation, superoxide anion generation and aggregation (Korchak et al., 1984). In this context, 52 the physiological relevance Of PCB-induced increases in [Ca2+]; is not only associated with neutrophil activation but can be important in terms Of impairment Of neutrophil function. Depletion Of intracellular Ca2+ stores by PCBs may lead tO a decrease in neutrophil response when a subsequent stimulus is applied. For instance, it is known that reduction Of [Ca2+]i resulted in abrogation Of neutrophil activation by macrophage-derived neutrophil chemotactic factor produced after Japanese encephalitis virus infection in mice (Srivastava et al., 1994). Experimental evidence suggests that Influenza A virus deactivates neutrophils via an increased resting [Ca2+], (Hartshorn et al., 1988). Furthermore, the requirement for extracellular Ca2+ during neutrophil phagocytosis (Sawyer et al., 1985) suggests that PCBs could impair bacterial clearing given the blockage by Aroclor 1242 Of Ca2+ influx in fMLP-treated neutrophils. Thus, taken together the results presented here suggest that Aroclor 1242 mobilizes Ca2+ from IP3-sensitive intracellular stores and also blocks Ca2+ influx following IP3R-mediated depletion Of intracellular stores. These events can have implications in the activity Of the immune system tO battle pathogens. 53 Chapter III ROLE OF PROTEIN PHOSPHORYLATION IN ACTIVATION OF PHOSPHOLIPASE A2 BY THE POLYCHLORINATED BIPHENYL MIXTURE AROCLOR 1242 54 III.1. Summary Polychlorinated biphenyls (PCBs) activate neutrophils tO induce degranulation and undergo superoxide anion production through a mechanism that involves stimulation Of phospholipase A2 (PLAZ). Since the biochemical processes leading to the PCB-induced activation Of PLAZ are unknown, the Objective Of this study was to determine whether or not protein phosphorylation, a common intracellular pathway, has a role in this mechanism. Isolated, rat neutrophils were labeled with [3H]-arachidonic acid ([3H]-AA), and activation Of PLA2 was determined from release Of radioactivity into the medium. Exposure tO the PCB mixture Aroclor 1242 induced release Of [3H]-AA, and pretreatment with bromoenol Iactone (BEL), an inhibitor Of Ca2+-independent PLA2, diminished release by 80%. Genistein, an inhibitor Of tyrosine kinases, caused a small but significant decrease in Aroclor 1242-stimulated release Of [3H]-AA. Daidzein, a genistein analog with nO activity to inhibit tyrosine kinases, had no effect on [3H]-AA release. An inhibitor Of p38 mitogen-activated protein kinase (MAPK), 88203580, did not affect Aroclor 1242-induced PLA2 activity at concentrations selective for p38 MAPK; however, PD 98059, which inhibits MAPK kinase (MEK), decreased [3H]-AA release to about the same extent as genistein. Treatment Of neutrophils with Aroclor 1242 induced phosphorylation Of p44 MAPK, and this phosphorylation was unaffected by 55 BEL but was inhibited by PD 98059. Staurosporine, a non-selective inhibitor Of protein kinase C (PKC), inhibited PCB-induced release Of [3H]- AA. RO 32-0432, a selective inhibitor of PKC, and PKCB1, produced the greatest degree Of inhibition (40%) among the tested protein kinase inhibitors. These results suggest that tyrosine kinases, PKC, and the MEK/MAPK pathway are involved in a fraction Of Aroclor 1242-induced activation Of PLA2. Ill. 2. Introduction Polychlorinated biphenyls are widespread, persistent chemicals present in almost all the ecosystems and trophic levels. Theoretically, chlorine substitutions in the aromatic ring lead tO the existence Of 209 congeners. Congeners with chlorine substitution in the ortho position are termed non-coplanar, whereas congeners without ortho chlorines are considered coplanar PCBs. This chemical characteristic defines many Of the toxicological profiles Of PCBs as non-dioxin-like or dioxin-like for orthO- and non-orthO-substituted congeners, respectively. In humans, about 130 individual PCB congeners have been detected in blood, milk and other tissue samples (Geisy and Kannan, 1998). Of interest is the fact that on‘hO-chlorinated PCBs are the most commonly found in human tissue after environmental or occupational exposure (Korrick and Altshul, 1998). 56 The toxicology Of PCBs has been extensively studied worldwide in diverse species. Some Of the effects that have been attributed to PCBs include neurotoxicity (Choksi et al., 1997; Seegal, 1999; Rice 91‘ al., 1999; Hany et al., 1999b), endocrine disruption (Desaulniers et al., 1999; KatO et al., 1999; Gould et al., 1999), impairment Of reproduction (Hugla and Thome, 1999), immunosuppression (Chang et al., 1982; Arnold et al., 1999; Wu et al., 1999), and cancer-related pathologies (Moysich et al., 1999; Nakanishi et al., 1999), although there is controversy around this latter effect (Dorgan et al., 1999). Immunosuppression has been one Of the most studied and polemical toxic effects Of PCBs (Lahvis et al., 1995; Harper et al., 1995; Chang et al., 1982). The presence Of these contaminants in different species has been associated with impairment Of non-specific immunity, including changes in natural killer function (De Swart et al., 1996) and neutrophil counts (Wu et al., 1999), and detriment Of macrophage phagocytosis (Ville et al., 1995). A well known effect Of PCBs in the immune system is the activation Of neutrophils (Ganey et al, 1993). Exposure in vitrO tO the PCB mixture Aroclor 1242, which contains mostly orthO-chlorinated biphenyls (Geisy and Kannan, 1998), activates several intracellular signals in neutrophils including phospholipase A2 (PLA2), tyrosine kinases (TKs) and phospholipase C (PLC) (Tithof et al., 1997; Tithof et al., 1998, Tithof et al., 1995). The interdependence Of these 57 pathways in PCB-induced neutrophil stimulation is not known. One Of the early events during exposure Of neutrophils tO PCBs is the activation Of a Cay-independent PLA2 (iPLAz), and inhibition Of this enzyme abrogates PCB-induced production Of superoxide anion by the cells (Tithof et al., 1998) In general, PLA2 releases arachidonic acid (AA) from membrane phospholipids tO function as a second messenger and a substrate for cyclooxygenases and Iipoxygenases. Several isoforms Of PLA2 have been identified (See Table l.1), raising questions about the specific functions Of each Of them. Among its functions, iPLAz participates in phospholipid remodeling by regulating the incorporation Of AA into membrane phospholipids by providing the Iysophospholipid acceptor employed in the acylation reaction (Balsinde et al., 1995). iPLAz can exist both in the cytosol (Hazen et al, 1990) and associated with membranes (Hazen et al., 1991). The regulation Of this isoform Of PLA2 is incompletely understood; however, ATP has been identified as one Of the regulators Of its enzymatic activity (Hazen and Gross, 1991; Ma et al., 1998). Furthermore, iPLA2 can be found forming complexes with other proteins such as phosphofructokinase (Hazen and Gross, 1993) and calmodulin (Wolf and Gross, 1996), which may represent other levels Of regulation. Given that iPLAz plays an important role in neutrophil stimulation by PCBs, it is Of interest to understand its regulation during exposure tO 58 PCBs. Protein kinases also play a critical role in PCB-induced activation Of neutrophils (Tithof et al., 1997), therefore we began exploring whether activation Of iPLAz depends on phosphorylation pathways. Accordingly, the purpose Of the present study was to examine the effects Of pharmacological intervention Of phosphorylation cascades on activation Of iPLA2 by PCBs. III. 3. Materials and methods III. 3. A. Chemicals PCBs were purchased from ChemService (West Chester, PA). BEL was purchased from BiomOI (Plymouth Meeting, PA). [3H][5,6,8,9,11,12,14,15]-AA ([3H]-AA; 180-240 Ci/mmol) was purchased from DuPont NEN (Boston, MA). Genistein and daidzein were Obtained from Sigma Chemical Company (St. Louis, MO). PD 98059, SB 203580, staurosporine, and RO 32-0432 were purchased from Calbiochem (San Diego, CA). Concentrations Of inhibitors were selected based on literature reports Of effective concentrations and low cytotoxicity. III. 3. B. Isolation Of rat peritoneal neutrophils Neutrophils were isolated from the peritoneum Of male, Sprague—Dawley, retired breeder rats by glycogen elicitation (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt 59 solution (HBSS), pH 7.35, containing 1.6 mM CaClz. The percentage Of neutrophils in the cell preparations was > 95%, and the viability was >95% determined by the ability tO exclude trypan blue. The isolation procedure was performed at room temperature. III. 3. C. Exposure tO PCBs PCB stock solutions were prepared by dissolution in N,N- dimethylformamide (DMF). Neutrophils (2x106) were suspended in HBSS (1 mL) in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 uL Of the PCB stock solution was added tO the tubes tO achieve the desired concentration. Control neutrophils received 1 uL Of DMF. III. 3. D. Determination Of PLAz activity Neutrophils (107/mL) were suspended in Mg?”- and Ca2+-free HBSS containing 0.1% bovine serum albumin and incubated in the presence Of 0.5 uCi/mL [3H]-AA for two hours, gently shaking at 37°C. Neutrophils were then washed twice with Mg?”- and Ca2+-free HBSS. The cell count was adjusted so that the final concentration Of neutrophils was 2x106/mL. Total cellular uptake Of [3Hj-AA was measured in a 1-mL aliquot Of suspended cells: the incorporation Of [3H]-AA was routinely between 80-88%. PLAZ activity was measured as the release Of [3H]-AA from labeled neutrophils (Tithof et al., 1998) incubated with inhibitors for 20 minutes (37°C) and then 60 exposed tO Aroclor 1242 at 37°C for 30 min, unless otherwise stated. At the end Of each incubation, neutrophils were placed on ice and spun in a centrifuge at 0°C for 10 min. The cell-free supernatant fluids were transferred tO vials containing scintillation cocktail (14 mL), and the total radioactivity in each sample was determined by liquid scintillation counting. Ill. 3. E. Detection of phosphorylated p42/p44 MAPKs Neutrophils (3 x 10° /mL) were suspended in HBSS and incubated with inhibitors for 20 min at 37°C before stimulation with 10 ug/mL Aroclor 1242 or vehicle and incubated for an additional 30 min at 37°C. After incubation, samples were spun in a centrifuge at 4°C for 10 min. The pellets were washed twice with phosphate-buffered saline (PBS) (pH 7.4) and resuspended in 300 pL lysing buffer (20% sodium dodecyl sulfate, SDS) for one hour and then sonicated for 30 sec. Samples were denatured by boiling for 5 min at 100°C and separated on a denaturing, 10% SDS polyacrylamide gel. Approximately 50 pg Of protein were added to each lane. Proteins were transferred electrophoretically to nitrocellulose membranes. After transfer, membranes were blocked for 3-4 hours in Tris-buffered saline (TBS) + Tween 20 (1%) (TBS-T) containing 4% chicken ovalbumin and 0.25% sodium azide. Membranes were incubated with mouse anti-phosphorylated MAPK antibody (New England BiOLabs) (1:2500) in blocker for two hours with constant rocking. Blots were washed three times with TBS-T (30, 5 and 61 5 min) and once with TBS (5 min). Goat anti-mouse IgG linked to horseradish peroxidase (1:7500) in TBS was added for 1 hour. Blots were washed using the same protocol as described above. Enhanced chemiluminiscence using Amersham reagents was performed tO visualize labeled, phosphorylated proteins. III. 3. F. Cytotoxicity assay Release by neutrophils Of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium was used as an indicator Of cytotoxicity. The method for sample preparation was the same as that described above. LDH activity present in the supernatant fluid was measured according to the method Of Bergmeyer and Bernt (1974). III. 3. G. Statistical analysis Data are presented as the mean a SEM from at least 4 different experiments conducted in triplicate. TO calculate percent Of control for release Of [3H]-AA in studies using inhibitors, first the values for release Of [3H]-AA in the absence of Aroclor 1242 at each concentration Of inhibitor (including vehicle control) were subtracted from the corresponding values in the presence Of Aroclor 1242 to calculate the specific Aroclor 1242-induced release. Second, the value for Aroclor 1242-induced release in the absence Of inhibitor (vehicle control) was taken to be 100%. Angular transformation 62 (arcsin) was used on percentage data to generate an approximated gaussian distribution. Detection Of significant differences among treatments was determined using analysis Of variance (ANOVA) and Tukey test as a post hoc test. When angular transformation did not produce gaussian data, Kruskal Wallace ANOVA on Ranks was used. Two-tailed p-values <0.05 were considered significant. III. 4. RESULTS III.4.A. Aroclor 1242 induced release Of [3H]-AA from neutrophils ' The time course Of cumulative release Of [3H]-AA (ie, PLA2 activity) induced in rat neutrophils by 10 ug/mL Aroclor 1242 is presented in Figure |II.1A. [3H]-AA release increased exponentially for the first 30 min, after which little additional [3H]-AA was Observed. Based on this graph, all the experiments measuring PLA2 activity were conducted using a 30-min incubation period. An important feature Of iPLA2 is that it can be selectively, irreversibly inhibited by BEL (Hazen et al., 1991) with 1000-fold greater specificity than for Ca2+-dependent PLA2. BEL inhibited Aroclor1242-induced 63 g 40 ,A O Aroclor1242 I- O DMF "5 s: 30- I § i 1 2 201 a: a: :t 10- 0 co: fit“) 0 7° 0 0 30 60 90 120 150 180 Time (min) 3120 B 8100- 0 “6 a: 80- $ 60- m 2 g; 40- 5.20. ”E“ -- 0 0.01 0.1 i 1'0 1'00 BEL (pM) Figure "M. A. Time-course for Aroclor 1242-induced release of [3H]-AA from labeled neutrophils. B. Dose-response curve for BEL-induced inhibition Of Aroclor 1242- elicited release Of [’H]-AA. Cells were exposed to 10 ug/mL Aroclor 1242, and release Of [3H]-AA was determined as described in Materials and Methods. Data are expressed as means i SEM Of four different experiments with triplicates per assay. 64 release Of [3H]-AA (Figure III.1.B), consistent with previous results (Tithof et al., 1998). At 100 (M BEL, total inhibition was not achieved. III.4.B. Involvement Of protein kinase pathways in Aroclor 1242-induced activation Of PLA2 Aroclor 1242 induces a rapid protein tyrosine phosphorylation in neutrophils (Tithof et al., 1997). Furthermore, either inhibition Of tyrosine kinase with genistein or inhibition Of iPLA2 with BEL diminished production Of superoxide anion by Aroclor 1242-treated neutrophils (Tithof et al., 1997, 1998). It was Of interest to examine whether these pathways are interrelated; ie, is iPLAz activation dependent on tyrosine kinase activity? Genistein significantly inhibited Aroclor 1242-induced PLA2 activity in a dose-dependent manner (Figure Ill.2) with a maximal inhibition Of approximately 20%. After 5 min exposure to Aroclor 1242, inhibition was Observed with concentrations Of genistein 2 1 uM. After 30 min Of Aroclor 1242 exposure larger concentrations Of genistein (2 50 nM) were required to inhibit a fraction Of the release Of [3H]-AA. The concentrations Of genistein which produced significant inhibition also blocked respiratory burst in fMLP-stimulated neutrophils (Combadiere et al., 1993) and are consistent with those reported to inhibit phosphorylation Of cPLA2 (Ambs et al., 1995). Daidzein, a genistein analog without effect in tyrosine phosphorylation, had no significant effect on release Of [3H]-AA in 65 140 120 - 80- + Genistein * —I— Daidzein 4o . . . . 0.01 0.1 1 10 100 Drug (PM) 63 O [3H]-AA Release (% Of control) Figure Ill.2. Dose-response curve for inhibition of Aroclor 1242 (10 ,ug/mL)- induced release Of[3H]-AA by Genistein or Daidzein. Cells were incubated with the indicated concentrations Of Genistein or Daidzein as described in Materials and Methods. Data are expressed as means 3: SEM Of four different experiments with triplicates per assay. *, p<0.05 vs. [3H]-AA released by neutrophils exposed to 10 ug/mL Aroclor 1242 alone. 66 Aroclor 1242-treated neutrophils. These results suggest that tyrosine phosphorylation is responsible for a fraction Of Aroclor 1242-induced PLA2 acfivafion. TO investigate the possible involvement Of a phosphorylation cascade leading to PLA2 activation induced by Aroclor 1242, the effects Of SB 203580, an inhibitor Of p38 mitogen activated protein kinase (MAPK), and PD 98059, a MAPK kinase (MEK) inhibitor, were examined. At concentrations below 50 pM, SB 203580 did not affect Aroclor 1242- induced [3H]-AA release (Figure III.3.A). At 50 and 100 (M, SB 203580 caused about a 20% decrease in release Of [3Hj-AA. PD 98059 caused a small but significant decrease in [3H]-AA release at 1 pM (Figure III.3.B), but the extent Of inhibition at larger concentrations (50 and 100 nM) was about 20% (Figure III.3.B). These results suggest that a MAPK pathway involving MEK and p42/p44 MAPK is important for a fraction Of the AA release Observed upon stimulation Of neutrophils with PCBs. TO examine the interdependence Of PCB-induced activation Of PLA2 and MAPK, Western immunoblotting assays were conducted. Aroclor 1242 induced the phosphorylation Of p44 MAPK, commonly referred tO as extracellular signal-regulated kinase (ERK-1), and increased the phosphorylation Of the constitutively expressed, phosphorylated p42 MAPK (ERK-2) (Figure III.4). This event was insensitive tO BEL but was blocked by PD 98059. 67 / A A O N O \ O ...__§ * m C N5 00 O \\ [3H]-AA Release (% Of Control) a, o 0 “1 i ""”'1'0 ' "50"1'00 SB 203580 (pM) 100 - 90 i 80 - 70 - 60 - 50 - 40 [3H]-AA Release (% Of control) 0.0 0.01 0.1 i 10 100 PD 98059(|.lM) Figure |II.3. A. Dose-response curves for (A) SB 203580- and (B) PD 98059- induced inhibition Of Aroclor 1242 (10 ,ug/mL)-stimulated release of fHJ- AA. Cells were incubated with the indicated concentrations Of SB 203580 or PD 98059 as described in Materials and Methods. Data are expressed as means i SEM Of four different experiments with triplicates per assay. *, p<0.05 vs. [3H]-AA released by neutrophils exposed to 10 pg/mL Aroclor 1242 alone. 68 44kD .. 4 ._.... 42k023~5 . 8 .- DMF Aroclor BEL PD 98059 + + Aroclor Aroclor Figure III.4. Aroclor 1242-stimulated MAPK phosphorylation in rat neutrophils and effects of BEL and PD 98059. Cells were treated with DMF or with Aroclor 1242 (10 ug/mL) in the absence or presence Of BEL (10 uM) or PD 98059 (50 nM). Phosphorylated MAPKs were detected as described in Materials and Methods. Results are representative Of two separate experiments. 69 In BEL-treated neutrophils, treatment with either genistein or PD 98059 increased slightly the inhibition Of [3H]-AA release (63% for either genistein plus BEL or PD 98059 plus BEL vs 56% with 10 uM BEL alone). This minor increase in inhibition suggests that genistein and PD 98059 inhibit the same pathway as BEL, providing further support for the hypothesis that tyrosine kinases contribute in part tO the activation Of iPLA2. Although Western analysis does not measure activity, increased phosphorylation is in general accompanied by increased activity (Hashimoto et al., 1999; Qiu and Leslie, 1994) so that these results imply that Aroclor 1242 increased activity Of p44 MAPK. Staurosporine, a non-specific PKC inhibitor, slightly increased [3H]- AA at 20 nM and had no effect at concentrations between 20 and 500 nM; however, at 1 pM it decreased Aroclor 1242-induced release Of [3H]-AA by 35% (Figure lll.5.A). RO 32-0432, a highly specific inhibitor Of PKC0L and PKCm (Wilkinson et al., 1993), decreased release Of [3H]-AA in a dose- related manner with significant inhibition (s 40%) at 5-10 uM (Figure lll.5.B). RO 32-0432 also increased inhibition Of [3H]-AA release in BEL- treated neutrophils (80% vs. 56% BEL alone). These data suggest that PKC may be involved not only In iPLA2 activation but also in the mechanism which accounts for the residual [3H]-AA release not sensitive to BEL. 70 AAA N-thON-h OOOOOOO [3H]AA Release (% or Control) O 3;. 0‘ 10 ' 100' ""1660 Staurosporine (nM) 110 100 i B 90 - 80 - 70 - 60 . 50 . 40 [3H]AA release (%Of control) 0.001 0.01 0:1 1 10 Ro 32-0432 (pM) Figure II|.5. Dose-response curves for (A) Staurosporine- and (B) R0 32- 0432-induced inhibition Of Aroclor 1242 (10 pg/mL)-stimulated release Of [3H]-AA. Cells were incubated with the indicated concentrations Of Staurosporine or Ro 32-0432 as described in Materials and Methods. Data are expressed as mean i S.E.M Of four different experiments with triplicates per assay. *, p<0.05 vs. [3H]-AA released by neutrophils exposed to 10 ug/mL Aroclor 1242 alone. 71 III. 5. Discussion Previous studies have shown that Aroclor 1242-mediated neutrophil activation involves the participation Of PLA2 and tyrosine kinases (Tithof et al., 1998; Tithof et al., 1997). New findings reported here suggest that part Of the Aroclor 1242-induced PLA2 activity is dependent on a phosphorylation cascade involving MEK, and that Aroclor 1242-induced phosphorylation Of MAPK is independent from the activation Of iPLAz. Inhibition Of iPLAz activity by BEL was maximal at 50 pM (Fig ”H B), which is consistent with saturation Of inhibition Observed by Lehman et al., (1993). The maximal inhibition Of [3H]-AA release was 80-90%; the remaining 10-20% Of [3H]-AA release might be associated with the activation Of other phospholipases including Ca2+-dependent isoforms Of PLA2 which are not sensitive to BEL at these concentrations. It has been demonstrated previously that inhibition Of tyrosine kinase with genistein diminished production Of superoxide anion by Aroclor 1242- treated neutrophils (Tithof et al., 1997). Similarly, BEL inhibited Aroclor 1242-stimulated superoxide anion generation (Tithof et al, 1998). It was Of interest tO examine whether these pathways are interrelated; ie, is iPLA2 activation dependent on tyrosine kinase activity? Treatment with genistein reduced [3H]-AA release induced by Aroclor 1242 by about 20% (Fig. 2), suggesting that tyrosine kinases may play a role in activation Of PLA2. 72 MAPKs, particularly p38 MAPK, are activated by tyrosine phosphorylation (Krump et al., 1997), and a MAPK activity dependent on tyrosine phosphorylation has been Observed in fmlp-activated neutrophils (Torres et al., 1993). Both p38 MAPK (Borsch et al., 1998) and ERK1/ERK2 (Nemenoff et al., 1993, Milella et al., 1997) can directly phosphorylate and activate the cytosolic PLA2 (cPLAg). The use Of SB 203580 and PD 98059 has been considered a powerful tOOl tO differentiate between different MAPK activities (Gould et al., 1995). SB 203580 is a potent and selective inhibitor Of p38 MAPK, whereas PD 98059 inhibits phosphorylation Of ERK1/ERK2 (Zu et al., 1998). SB 203580 specifically abolishes the enzymatic activity Of p38 MAPK (Ridley et al., 1997) at concentrations as low as 1-10 uM (FOItz et al., 1997; Cuenda et al., 1995). The SB 203580 concentrations that significantly decreased Aroclor 1242-induced PLA2 activity (50-100 (M) (Fig. 3A) presume a minor role for p38 MAPK in this response; however, a total lack Of participation Of p38 MAPK cannot be ruled out from these data. In contrast to SB 203580, inhibition Of [3H]-AA release was Observed with concentrations Of PD 98059 selective for MEK (Alessi et al., 1995). The dose-response curve Observed for PD 98059 suggests that MAPK is involved in about 20-25% Of [3H]-AA released during Aroclor 1242- induced activation Of PLAz (Fig. 3B). The lack Of inhibition Of Aroclor 1242- induced phosphorylation Of p44 MAPK by BEL (Fig. 4) also indicates that 73 activation Of this MAPK occurs upstream Of activation Of PLA2. Tyrosine kinase activation probably occurs prior tO stimulation Of PLA2 as well because genistein inhibited [3H]-AA release. Activation Of tyrosine kinases may contribute to activation Of ERK1/ERK2 as has been described (Peavy and Conn, 1998; Luconi et al., 1998). Interestingly, the MEK pathway can be activated through ras proteins including H-ras, K-ras, N-ras, and R-ras (Waddick and Uckun, 1998), and the function Of these proteins has been implicated in the etiology Of large variety Of human tumors (Beaupre and Kurzrock, 1999; Kato et al., 1999). If these same pathways activated by Aroclor 1242 in neutrophils are also involved in other cell types and if a ras- dependent pathway is involved in Aroclor 1242-induced cell activation, it may explain some Of the reported associations between orthO-chlorinated biphenyls and cancer (Gribaldo et al., 1998; Haag et al., 1997) and promotion activity Observed for some PCB mixtures (Nakanishi et al., 1999). For instance, it has been suggested that PLA2 activity is increased in colorectal tumors (Hendrickse et al., 1995). Moreover, it is known that inhibition Of the MEK pathway can suppress as much as 80% Of tumor growth in mice (Sebolt et al., 1999; Seufferlein et al., 1999). Thus, it may be that activation Of both PLAZ and the MEK pathway induced by Aroclor 1242 play an important role in some Of the effects Observed in other tissues following exposure to PCB mixtures. 74 Among the protein kinase inhibitors, the greatest degree Of inhibition Of Aroclor 1242-induced PLA2 activity was produced by the PKC inhibitors staurosporine and RO 32-0432, which suggests a key role for PKC (Figs. lll.5A and lll.5B). RO 32-0432 is highly selective for PKCO, and PKC,“ (Wilkinson et al., 1993), two Cay-dependent isoforms Of PKC (Lin and Chen, 1998). The effectiveness Of RO 32—0432 at blocking Aroclor 1242- induced activation Of PLA2 suggests that specific isoforms Of PKC may be critical to cell activation. These data are consistent with reports that Aroclor 1242 stimulates PKC in cerebellar granule cells (Kodavanti et a/., 1995) PLAZ activation can be triggered following activation Of the MEK/MAPK pathway (Milella et al., 1997; Wheeler et al., 1997) and in turn, the MEK pathway can be stimulated in a PKC-dependent manner (Zhang et al., 1998; Kumar et al., 1997; Slevin et al., 1998), and PKC activation can be initiated by tyrosine phosphorylation (Bradford and SOItOff, 1998). These Observations can be synthesized to a model involving the phosphorylation cascade: tyrosine kinase—>PKC—>MEK—>ERK (Aharonovitz et al., 1998; Muthusamy and Leiden, 1998) that might lead to subsequent phosphorylation and activation Of PLA2 (Huwiler et al., 1997). However, activation Of PLA2 by PKC can be independent from p42/p44 MAPK (Husain and Abdel, 1998). 75 The data presented here suggest that tyrosine kinase, PKC, MEK and ERK contribute tO a fraction Of total PLA2 activity induced by Aroclor 1242. An interesting possibility is that the phosphorylation cascade does not target PLA2 directly but indirectly through other proteins. For instance, MAPK (Mizutani et al., 1993) and PKC (Regnouf et al., 1995) can each phosphorylate annexins, proteins with known inhibitory activity on PLA2 (Mira el al., 1997; Haigler et al., 1987). Phosphorylation may initiate conformational changes or subcellular translocations leading tO disinhibition Of PLA2. An interesting question deals with the mechanism Of the initial activation Of the phosphorylation—dependent pathway. At least three possibilities can be considered. First, Aroclor 1242 may activate tyrosine kinases or PLA2 directly. PCBs have been shown to interact directly with PKC, inducing its activation and membrane translocation (Kodavanti et al., 1995; Kodavanti and Ward, 1998). In addition, Aroclor 1242 elicited activation Of PLA2 in cell lysates (Tithof et al., 1998), although it is unknown whether this was a direct activation Of the enzyme or occurred through other cytosolic factors. A second possibility is the direct activation Of a receptor by PCBs, leading tO enzyme phosphorylation. Receptor-mediated modulation Of iPLAz has been reported tO occur through a Gq/G11-coupled mechanism (Derrickson and Mandel, 1997). Although Gq can be coupled to activation Of PKC, tyrosine kinase and MAPK (Naor et al., 1998), it has 76 been reported that in neutrophils production Of superoxide activation is unlikely to proceed through a G protein-dependent mechanism (Tithof et al., 1997). Structure-activity relationships with PCBs suggest that such a receptor is not the Ah receptor, but its identity is unknown (Olivero and Ganey, 1998). There is some evidence, however, that PCBs interact with nerve growth factor receptors (Angus and Contreras, 1995), so the possibility for receptor interaction beyond the AhR exists. A third possibility may be related tO PCB-dependent autocrine activation Of neutrophils. Recently it has been reported that secretory PLA2 (sPLA2) can activate neutrophils (Zallen et al., 1998) and other cell types (Hernandez et al., 1998) as a ligand which interacts with specific receptors on the membrane surface and independently Of its enzymatic activity. Neutrophils store sPLA2 in granules and secrete it in response tO soluble stimuli (Rosenthal et al., 1995). Neutrophil degranulation after exposure tO PCBs occurs in a structure-dependent manner (Olivero and Ganey, 1998). Thus, PCBs may initially induce degranulation, releasing sPLAz which activates the phosphorylation cascade as described above. In summary, the PCB mixture Aroclor 1242 activates neutrophils through multiple mechanisms. PLA2 activation by Aroclor 1242 occurs through different signal transduction pathways involving tyrosine kinase, PKC, MEK and ERK1/ERK2. The above findings add a new aspect toward the recognition Of cellular targets for PCBs. 77 Chapter IV CALCIUM/CALMODULIN-DEPENDENT REGULATION OF PHOSPHOLIPASE A2 DURING NEUTROPHIL ACTIVATION BY POLYCHLORINATED BIPHENYLS 78 AI‘ '— IV.1. Summary The effects Of Ca2+ and Ca2+lcalmodulin on the polychlorinated biphenyl (PCB)-induced activation Of phospholipase A2 (PLA2) in rat neutrophils was examined. The commercial PCB mixture Aroclor 1242 induced activation Of PLA2 and promoted an increase in intracellular free calcium concentration ([Ca2+]i). Bromoenol Iactone (BEL), an inhibitor Of the CaZ‘lindependent PLA2 (iPLAz) isoform activated by PCBs, did not abrogate the increase in [Ca2+]i, suggesting that this change in [Ca2+], is not downstream from the activation Of iPLAz. TMB-8, a blocker Of the release Of intracellular Ca”, induced a small but significant decrease in Aroclor 1242-induced stimulation Of PLAZ in a dose-dependent manner with a maximal inhibition of 17% at 50 nM. These two results suggest little direct dependence between the PCB-induced activation Of iPLA2 and increase in [Ca2+]i. Trifluoperazine (TFP), W7 and calmidazolium, three chemically distinct calmodulin inhibitors, inhibited significantly Aroclor 1242-induced PLA2 activity in a dose-dependent manner. Moreover, both Aroclor 1242 and TFP blocked the degranulation induced by the chemotactic agent fMLP. These results suggest the possibility that Aroclor 1242 alters PMN function through interaction with calmodulin. 79 IV.2. Introduction The chemical stability Of polychlorinated biphenyls (PCBs) makes this group Of organochlorine compounds one Of the most ubiquitous and persistent chemicals in the environment. These chemicals have been detected around the world In remote areas (Fromberg et al., 1999), wildlife refuges (Guillette et al., 1999), dairy products (Ramos et al., 1999) and human breast milk (Czaja et al., 1999, Greizerstein et al., 1999). The multiple cellular mechanisms Of action, widespread environmental distribution, physicochemical properties and differences in activity among congeners have made the toxicological profile Of PCBs both extensive and complex. The physiological effects Of PCBs in mammals include behavioral impairment (Rice, 1999), stimulation Of oscillatory uterine contraction (Bae et al., 1999), changes in immunoglobulins M and G (Arnold et al., 1999), and enzyme induction (Lagueux et al., 1999). Some evidence has suggested a relationship between PCB exposure and effects on motor functioning (Schantz et al., 1999), neurotoxicity (Seegal et al., 1999), cancer (Dorgan et al., 1999) and cancer-derived mortality (Kimbrough et al,1999) The mechanisms Of action Of PCBs are multiple and structure- dependent. Many Of the PC83 can bind with relatively high affinity tO the aryl hydrocarbon (Ah) receptor and elicit toxic responses similar to those 80 Observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Another group Of PCBs has low affinity for the Ah receptor and exhibits different biological activities such as neurotoxicity (Kodavanti et al., 1997, Wong et al., 1997), stimulation Of insulin release (Fischer et al., 1996), and effects on neutrophil function (Olivero et al., 1998, Brown et al., 1995). With respect tO neutrophils, PCBs both activate these cells and alter activation in response to other stimuli (Olivero et al., 1998). In addition to alterations in cellular function, several intracellular signals are activated by PCBs in neutrophils including phospholipase A2 (PLA2), tyrosine kinases (TKs) and phospholipase C (PLC) (Tithof et al., 1997,Tithof et al., 1998, Tithof et al., 1995). PLA2 hydrolyzes phospholipids at the sn-2 position to release the second messenger arachidonic acid. This enzyme regulates neutrophil function by modulating superoxide anion production (Dana et al., 1998, Henderson et al., 1993) and degranulation (Smolen et al., 1980). Indeed, inhibition Of PLA2 prevents PCB-induced stimulation Of these cells (Tithof et al., 1998). In rat neutrophils, both Cay-dependent and Cay-independent isoforms Of PLAZ have been identified, and most Of the PCB-induced activity is attributed tO activation Of Cali-independent PLA2 (iPLAz) (Tithof et al., 1998). The biochemical mechanisms underlying the regulation Of iPLAz are not known. It has been shown that iPLA2 can be regulated by ATP (Hazen et al., 1991, Ma et al., 1998), nitric oxide (Gross et al., 1995) and peptides 81 such [Arg°] vasopressin (Lehman et al., 1997) and parathyroid hormone (Derrickson et al., 1997). An intriguing Observation is that iPLAz can be modulated by direct protein-protein interactions with biomolecules such as phosphofructokinase (Hazen et al., 1993) and calmodulin (Wolf and Gross, 1996) Given that iPLA2 plays an important role in neutrophil stimulation by PCBs, it was Of interest to determine whether activation Of this enzyme depends on other intracellular signals. The Objective Of this study was to examine the interaction among several signalling pathways involved in activation Of neutrophils by PCBs. In particular, the roles Of intracellular Ca2+ and calmodulin in activation Of PLA2 were explored. |V.3. Materials and methods IV.3.A. Chemicals Aroclor 1242 was purchased from ChemService (West Chester, PA). BEL was purchased from Biomol (Plymouth Meeting, PA). [3H][5,6,8,9,11,12,14,15]—arachidonic acid ([3H]-AA; 180-240 Ci/mmOl) was acquired from DuPont NEN (Boston, MA). FormyI-methionyl-Ieucyl- phenylalanine (fMLP), cytochalasin B and trifluoperazine were Obtained from Sigma Chemical Company (St. Louis, MO). TMB-8, W-7, and calmidazolium were bought from Calbiochem (San Diego, CA). Fura-2/AM was Obtained from Molecular Probes (Eugene, OR). 82 IV.3.B. Isolation Of rat peritoneal neutrophils Neutrophils were isolated from the peritoneum Of male, Sprague—Dawley, retired breeder rats by glycogen elicitation as described (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.35, containing 1.6 mM CaClz. The percentage of neutrophils in the cell preparations was > 95%, and the viability was >95% determined by the ability tO exclude trypan blue. The isolation procedure was performed at room temperature. IV.3.C. Exposure to PCBs PCB stock solutions were prepared by dissolution in N,N- dimethylformamide (DMF). Neutrophils (2x106) were suspended in HBSS (1 mL) in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 uL Of the PCB stock solution was added to the tubes tO achieve the desired concentration. Control neutrophils received 1 pL Of DMF. IV.3.D. Determination of PLAz activity Neutrophils (107/mL) were suspended in Mg”- and CaZI-free HBSS containing 0.1% bovine serum albumin and incubated in the presence Of 0.5 uCi/mL [3H]-AA for two hours, gently shaking at 37°C. Neutrophils were then washed twice with M921 and Ca2+-free HBSS. The cell count was 83 adjusted, so that the final concentration Of neutrophils was 2x10°lmL. Total cellular uptake Of [3H]-AA was measured in a 1-mL aliquot Of suspended cells: the incorporation Of [3H]-AA was routinely between 80-88%. Release Of [3H]-AA from labeled neutrophils was measured in cells incubated with inhibitors for 20 minutes (37°C) and then exposed to Aroclor 1242 at 37°C for 30 min, unless otherwise stated. At the end Of each incubation, neutrophils were placed on ice and spun in a centrifuge at 0°C for 10 min. The cell-free supernatant fluids were transferred to vials containing scintillation cocktail (14 mL), and the total radioactivity in each sample was determined by liquid scintillation counting. IV.3.E. Neutrophil degranulation Degranulation was measured by the release from neutrophils Of the enzyme myeloperoxidase (MPO). Neutrophils (2x10° cells/mL) were suspended in HBSS and pretreated with 5 ug/mL cytochalasin B for 5 minutes at room temperature. The quiescent neutrophils were then exposed tO PCBs or vehicle for 10 min at 37°C followed by incubation for an additional 10 min at 37°C with 100 nM fMLP. Cells were centrifuged at 4°C, and the cell-free supernatant fluids were collected. Activity Of MPO in the medium is determined using the method Of Henson et al. (1978). 84 IV.3.F. Intracellular Free Ca2+ Measurements Neutrophils (2.5 x 10° cells/mL) were labeled by incubation for 25 min at 37 °C with 5 pM Fura-2/AM in HBSS. After loading, cells were washed with HBSS, and the cell count was readjusted tO 2 x 10° cells/mL. Fluorescence emission at 505 nm was monitored at room temperature with constant stirring, using a dual wavelength spectrofluorometer system with excitation at 340 and 380 nm. The [Ca2+]i was calculated from fluorescence intensity readings using the following equation: [032+]:Kd*Q(R-Rmin)/(Rmax- R). R Is the ratio Of emission intensities at 340 and 380 nm excitation (340/380), Rmin is the 340/380 ratio under CaZ+-free conditions, Rmax is the ratio under saturating Ca2+ concentrations; Kd is the dissociation constant Of the Ca”: Fura-2 complex; and Q is the ratio Of the 380 nm fluorescence under conditions Of minimum and maximum [Ca2+] conditions (Shao et al., 1998). The equilibrium dissociation constant, Kd, was taken from literature, 224 nM (Kankaanranta et al, 1995a). Rmax and Rm,n values for each assay were determined from the fluorescence intensities in the presence Of 0.01% Triton X-100 or 10 mM EGTA, respectively. These two parameters did not change significantly in the presence Of Aroclor 1242. Fluorescence emission after excitation at 360 nm was monitored and in all studies conducted remained constant during data collection. Increases in resting [Ca2+]; evoked by agonists were detected by measunng the change in fluorescence ratio and comparing this to fluorescence ratio after exposure 85 with the vehicle N,N-dimethyllformamide (DMF). Typical values for quiescent neutrophils ranged from 50 tO 100 nM. PCBs were added to labeled cells 50 sec after the start Of data collection. PCB stock solutions were prepared by dissolution Of the neat chemical in DMF, and 1 uL/mL Of the respective stock solutions was added tO the cells to achieve the desired concentration in a final cell volume Of 3 mL. When BEL was used, labeled cells were pre-incubated with the inhibitor for 20 minutes at 37 °C before PCBs were added. IV.3.G. Cytotoxicity assay Release by neutrophils Of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium was used as an indicator Of cytotoxicity. The method for sample preparation was the same as that described above. LDH activity present in the supernatant fluid was measured according to the method Of Bergmeyer and Bernt (1974). IV.3.H. Statistical methods Data are presented as the means a SEM from at least 4 different experiments conducted in triplicate. TO calculate percent Of control for release Of [3H]-AA in studies using BEL, first the values for release Of [3H]- AA in the absence Of Aroclor 1242 at each concentration Of inhibitor (including vehicle control) were subtracted from the corresponding values in 86 the presence Of Aroclor 1242 to calculate the specific Aroclor 1242-induced release. Second, the value for Aroclor-induced release in the absence Of inhibitor (vehicle control) was taken tO be 100%. Angular transformation (arcsin) was used on percentage data tO generate an approximated gaussian distribution. Detection Of significant differences among treatments was determined using analysis Of variance (ANOVA) and Tukey test as a post hoc test. When angular transformation did not produce gaussian data, Kruskal Wallace ANOVA on Ranks was used (SigmaPlot, V4. Jandel Scientific. San Rafael CA. 1996). Two-tailed p-values <0.05 were considered significant. IV.4. Results IV.4.A. Involvement Of Cay/calmodulin in PCB-induced activation Of PLA2 It has been reported previously that PCB-induced activation Of PLA2 is independent Of extracellular Ca” (Tithof et al., 1998). TO investigate whether intracellular Ca2+ is important for Aroclor 1242-induced PLA2 activation, neutrophils were pretreated with the intracellular Ca” release blocker, TMB-8 (Kemmerich and Pennington, 1988). TMB-8 (25- 50 pM) produced a small but significant decrease in Aroclor 1242-elicited release Of [3H]-AA (Table IV.1). This suggests that a fraction Of the total 87 PLA2 activity induced by Aroclor 1242 depends on the release Of Ca2+ from intracellular stores. TMB-8 Concentration Release Of [3H]-AA from rat neutrophils (uM) (% control) 0 100 1 98.0.+..1.8 10 96.9i3.4 25 8713:363 50 83.3.Jc4.0a Table IV.1. TMB-8-induced inhibition of Aroclor 1242-stimulated release of [3H]-AA from rat neutrophils. Cells were preincubated with TMB-8 and then exposed to 10 ug/mL Aroclor 1242. Release Of [3H]-AA was determined as described in Materials and methods. Data are expressed as means i SEM Of four different experiments with triplicates per assay. a, Significantly different (p<0.05) from the control group (100%) when analyzed by ANOVA using Tukey test as a post hoc test. Exposure Of neutrophils tO Aroclor 1242 caused a time-dependent 2"]i (See Figure Il.1). This increase began after about 5 increase in [Ca minutes Of exposure tO PCBs and continued through 18 minutes. In order tO determine if this increase in [Ca2+]i was caused by the activation of PLA2 by Aroclor 1242, [Ca2+]i measurements were made in cells preincubated with the cell permeable PLA2 inhibitor BEL (10 nM). Treatment with BEL 88 did not abrogate the overall release Of [Ca2+]i elicited by 10 ug/mL Aroclor 1242 measured as area under the curve but produced a change in the kinetics Of the release over a period Of 18 minutes (Figure IV.1). Calmodulin is the main Ca2+ regulatory protein in eukaryotic cells. TO examine its role in Aroclor 1242-induced PLA2 activity the effects Of the calmodulin inhibitors, trifluoperazine (TFP), W7 and calmidazolium were determined. W7 induced a dose-dependent decrease in the Aroclor 1242- stimulated PLA2 activity with minimal cytotoxicity (Figure IV.2). TFP also caused a decrease in AA release, but only at significant cytotoxic concentrations (data not shown). About 50% inhibition was Obtained with calmidazolium, a very potent inhibitor Of calmodulin (IC50 s 0.05 nM) (Hait and Lazo, 1986). IV.4.B. TFP and Aroclor 1242-induced changes in neutrophil function Aroclor 1242 caused a dose-dependent inhibition Of fMLP- induced neutrophil degranulation (Figure IV.3.A) consistent with a previous report with orthO-chlorinated PCBs (Olivero et al., 1998). A similar effect Of TFP on neutrophil degranulation was Observed. Like Aroclor 1242, TFP blocked most Of the degranulation in response to fMLP (Figure IV.3.B). 89 Agonist BEL+DMF o I I I r I I 0 200 400 600 800 1000 Time (sec) Figure IV.1. Effects Of BEL on 10 pg/mL Aroclor 1242-induced increase in [Ca2+ ,- in rat neutrophils. Cells were loaded with fura- 2/AM as described in Materials and Methods. Fura-2-Ioaded neutrophils were incubated with 10 pM BEL for 20 min and then stimulated with 10 ug/mL Aroclor 1242 at the time indicated by the arrow. Data for control (DMF) are shown for comparison. Results are expressed as means 3r. SEM for four different experiments. 90 120 - -—100 IV 80 60 40 Percentage Of act 20 Control 5 10 20 40 1 TFP (pM) W7 (pM) CMDZ (pM) Figure IV.2. Effects of calmodulin inhibitors on phospholipase A; activity induced by 10 pg/mL Aroclor 1242. Neutrophils were labeled with [°H]-AA and incubated with different concentrations Of the calmodulin inhibitors TFP, W7 and calmidazolium (CMDZ) for 20 min at 37 °C followed by incubation with 10 ug/mL Aroclor 1242 for 30 min at 37 °C. The release Of [°H]-AA into the medium at each concentration was compared with the release by the same compound in absence Of the calmodulin inhibitor and presented as percentage Of activity. Results are expressed as means i SEM from four different experiments performed in triplicates. *, p<0.05 vs [3Hj—AA released by neutrophils exposed to 10 pg/mL Aroclor 1242 alone. 91 A 140 6' A .3: 120 n c O 0 100 - o..— O ,\° 80 - o g 60 - 2 q: a n: 40 O n- 20 "' E 0 A . . . 0.01 0.1 1 10 Aroclor 1242 (pg/m L) A 140 3 B 4': 120 c o . U 100 - b.— O °\° 80 - 3; 60 Cytotoxicity 8 ’ 7, 40 a: O 20 - a E 0 _ 0.001 0.01 0.1 1 10 100 TFP (pM) Figure IV.3. Inhibitory effect Of (A) Aroclor 1242 and (B) Trifluoperazine on fMLP-induced neutrophil degranulation. Cytochalasin-treated neutrophils were exposed to Aroclor 1242 or TF P for 10 min then incubated for another 10 min with 100 nM fmlp, and degranulation was measured as described in Materials and methods. Data are expressed as means i SEM Of four different experiments. 92 IV.5. Discussion PCBs cause an increase in [Ca2+]i in cerebellar granule cells (Shafer et al., 1996), hepatocytes (Atzori et al ., 1991), endothelial cells (Toborek et al., 1995) and human neutrophils (VOie et al.,1998). The mechanisms associated with this increase in intracellular Ca2+ are structure-dependent and, at least in neutrophils, have been associated with activation Of PLC (VOie and Fonnum, 1998). We have demonstrated that Aroclor 1242 increases PLA2 activity with release Of arachidonic acid (Tithof et al., 1998). Similarly, Aroclor 1242 induces an increase in [Ca2+], in rat neutrophils (Figure IV.1). One question addressed in this study is whether there is a causal relationship between activation Of PLA2 and the increase in [Ca2+],. For example, it is known that arachidonate (Rzigalinski et al., 1996) or its metabolites (Reynaud and Pace, 1997) can cause depletion Of intracellular calcium stores leading to a rise in [CaZ+]i. Following exposure Of neutrophils tO Aroclor 1242, arachidonic acid is released within 2 minutes (Tithof et al., 1998), whereas the increase in [Caz‘] begins after 5 minutes (Figure IV. 1). Given this it was hypothesized that inhibition Of PLA2 would block the Aroclor 1242-induced increase in [Caz’]. This was not the case (Figure IV.2). We have demonstrated previously that BEL abrogates Aroclor 1242-stimulated release Of arachidonate acid (Tithof et al., 1998), and the lack Of effect Of BEL on the 93 overall increase in [Ca2+]i elicited by Aroclor 1242 led us to conclude that PCBs act on [Ca2+]i through a signal that is not downstream from activation Of iPLA2; however, iPLA2 can modulate the kinetics of the process. This dissociation between iPLA2 and Ca2+ has also been Observed in aortic smooth muscle cells in which in the absence Of extracellular Ca2+ the spike in [Ca2+]i elicited by vasopressin was not abolished by treatment with BEL, however, in presence Of extracellular Ca”, BEL prolonged the duration Of the response (Lehman et al., 1993). In previous studies it has been demonstrated that the majority Of arachidonic acid released from neutrophils after PCB treatment comes from stimulation Of a CaZ+-independent PLAz and that this activation is not abrogated by the intracellular Ca2+ chelator BAPTA-AM (Tlthof et al., 1998). Studies presented here demonstrated that TMB-8 caused a small reduction Of Aroclor 1242-induced release Of arachidonic acid. This suggests that release Of Ca2+ from intracellular stores contributes to a fraction Of the PCB-stimulated PLA2 activity. Moreover, these results presume that BAPTA/AM and TMB-8 target different intracellular Ca2+ pools. It has been reported that TMB-8 inhibits fMLP- and PMA—stimulated superoxide anion production in neutrophils, and that these responses can be restored upon Ca2+ loading with A23187 (Weisdorf and Thayer, 1989), supporting that effects Of TMB-8 are due tO interference with normal Ca2+ responses. It has been suggested that TMB-8 causes its effects on 94 intracellular Ca2+ pools that may be associated with cytoskeletal elements that sequester intracellular Ca2+, or on lysosomal or other intracellular pools (Weisdorf and Thayer, 1989; Khan et al., 1985). Despite the Observation that the PCB-induced increase in [Ca2+]i is independent Of PLAZ activity (Figure NZ), and that the stimulation Of PLAZ by PCBs is largely independent Of Ca2+, there appears to be an interaction between Ca2+ signaling and release Of [3Hj-AA. This interpretation is based on the known relationship between Ca2+ and calmodulin and the results Of studies with TFP, W7 and calmidazolium in which these calmodulin inhibitors diminished PCB-stimulated release Of AA (Figure IV. 3). One explanation for these results is that calmodulin may regulate the iPLAZ activated by PCBs in neutrophils. Activity Of some enzymes is inhibited by calmodulin. For instance, in Limulus, Ca2+lcalmodulin-binding peptides and calmodulin itself block PLC activity, probably through a calmodulin-like structure present in the amino-terminal domain Of PLC (Richard et al., 1997). The fact that calmidazolium strongly blocks the activation Of iPLA2 by Aroclor 1242 suggests the participation Of calmodulin as regulator Of iPLAz activity. TFP, which also inhibited PCB-increased PLA2 activity by ~15%, binds tightly to calmodulin in solution as determined by nuclear magnetic resonance studies (Craven et al., 1996; Vandonselaar et al., 1994). TFP and W7 binding tO calmodulin causes a change in tertiary structure from an elongated dumb bell with exposed hydrophobic surfaces 95 to a compact globular form which can no longer interact with its target enzymes (Vandonselaar ef al., 1994; Ozawa et al., 1999) Regulation Of neutrophil iPLAz might be similar tO that Observed in myocytes, in which iPLA2 forms a complex with calmodulin, and this binding inhibits activity ONOIf and Gross, 1996). If this model Of enzyme regulation by calmodulin applies to neutrophil iPLA2, then PCBs may activate neutrophil iPLAz through interaction with calmodulin. The rank order Of potency for the inhibition Of calmodulin appeared to be calmidazolium > TFP > W7 (Ambudkar et al., 1989), which is consistent with the results presented here. TFP, W7 or calmidazolium may alter the conformation Of calmodulin such that the binding site for Aroclor 1242 is inaccessible. Alternatively, PCBs may activate iPLAz through a different mechanism, but inhibition Of calmodulin by TFP, W7 or calmidazolium interferes with this mechanism indirectly. For instance, it has been shown that W7 inhibits fMLP stimulation Of PLD in neutrophils with an IC50 value Of z 50 (M (Takahashi et al., 1996). Similarly, it was recently reported that the PCB-stimulated release Of insulin from RINm5F cells was blocked by an inhibitor Of Ca2+lcalmoduIin-dependent kinase (Fischer et al., 1999) suggesting that calmodulin is required for PCB-mediated signaling. Both TFP and Aroclor 1242 inhibited fMLP-induced neutrophil degranulation (Figure IV.4). The dose-response curve Obtained for TFP- elicited inhibition Of fMLP-induced neutrophil degranulation measured as 96 MPO release is very similar to that previously reported for the enzyme I3- glucuronidase (Smith et al., 1981), another marker Of neutrophil degranulation. Remarkably, 10 pM TFP elicited approximately 50% inhibition Of fMLP-induced neutrophil degranulation; this same concentration has been reported tO inhibit calmodulin activity (Hait et al., 1986). Although, TFP is cytotoxic at concentrations greater than 10 (M, the lack Of massive release Of MPO by this compound suggests that it targets the extracellular membrane and not the granule membrane. TFP interacts with annexin proteins and is capable Of inhibiting annexin l- and annexin II-mediated aggregation Of Iiposomes by releasing them from the plasma membrane (Blackwood et al., 1995). In addition, TFP blocks annexin |l tetramer—mediated membrane fusion (Liu et al., 1997). Annexins link secretory vesicles to the plasma membrane (Klee et al., 1998) and are linked tO calmodulin. Remarkably, calmodulin binds tightly to vacuoles during the post-docking phase Of vacuole fusion promoting bilayer mixing (Peters et al., 1998). Thus, this activity provides a potential mechanism by which TFP may inhibit degranulation Of neutrophils through calmodulin- dependent processes. Another possibility that may link the inhibition Of fMLP-induced degranulation tO an inhibitory effect Of TFP or PCBs on calmodulin is that membrane fusion requires the phosphorylation Of trafficking proteins such as syntaxin 3, a process proposed tO be mediated by Ca2+- and 97 calmodulin-dependent protein kinase II (Risinger et al., 1999). In consequence, the binding Of PCB to calmodulin would reduce the activity Of the calmodulin-dependent kinase and abrogate the degranulation process. These effects Of TFP and PCBs on fMLP-induced degranulation are not the only analogy between these two compounds. In neutrophils, Aroclor 1242 has been found tO block Ca2+ after depletion Of intracellular stores (See Chapter I). Similarly, calmodulin inhibitors block the same effect in several cell types (Mene et al., 1996; Haverstick et al., 1998). TO date, nO information is available regarding the interaction between PCBs and calmodulin. However, it has been proposed that one essential feature for Optimal ligand binding to calmodulin is the presence Of two hydrophobic, aromatic rings, such as is present in PC83 (Halt et al., 1986). From these results, it is evident that calmodulin might be one relevant target that mediates cellular responses elicited by PCBs. In summary, the PCB mixture Aroclor 1242 activates neutrophils through different signal transduction pathways involving CaZ+/calmodulin- dependent processes. Aroclor 1242 mimics the inhibitory properties Of the calmodulin inhibitor TFP on fMLP-induced degranulation. Aroclor 1242- induced release Of arachidonic acid via iPLAz is not coupled to the 2+It increase in [Ca elicited by Aroclor 1242. The above findings add a new aspect toward the recognition Of cellular targets for PCBs. 98 Chapter V BIOCHEMICAL SIGNALS INVOLVED IN THE ACTIVATION OF PLAz BY 2,2’,4,4’-TETRACHLOROBIPHENYL 99 V.1. Summary The PCB mixture Aroclor 1242 stimulates neutrophils to produce superoxide anion and release different enzymes. The activation Of neutrophils by PCBs require the cooperation Of diverse enzymes, particularly phospholipase A2 (PLAZ). The Objectives Of this study were to determine whether or not the stimulation Of PLA2 by Aroclor 1242 was shared by 2,2’,4,4’-tetrach|orobiphenyl, an orthO-chlorinated PCB, and investigate the mechanisms leading to its activation. Rat neutrophils were labeled with [3H]—arachidonic acid ([3H]-AA), and stimulation Of PLA2 was measured from release Of radioactivity into the medium. Exposure tO the orthO-PCB congener 2,2’,4,4’-tetrachlorobiphenyl, but not tO the non-orthO congener 3,3’,4,4’-tetrachlorObiphenyl induced a dose-dependent activation Of PLA2 and phosphorylation Of cPLA2 and p44 MAPK. About 50% Of the PLA2 activity was sensitive to BEL, an inhibitor Of Ca2+- independent PLAz, Pharmacological intervention using inhibitors showed that tyrosine kinases, ras, protein kinase C (PKC) and p42/p44 MAPK contributed to a fraction (20%) Of the total activity induced by 2,2’,4,4’- tetrachlorobiphenyl. In addition, the calmodulin inhibitors W7 and calmidazolium blocked up tO 40% Of the total activity. These results suggest that the orthO-chlorinated PCBs activate PLA2 through mechanisms involving tyrosine kinases, PKC, ras, the MAPK pathway and calmodulin. 100 v.2. Introduction Polychlorinated biphenyls are man-made chemicals that disrupt function Of a variety Of cells by interfering with multiple biochemical mechanisms. In the last two decades the importance Of these compounds has grown considerably due to their detection in human tissues (Angulo et al., 1999; Stewart et al). These contaminants are normally found in complex mixtures having both OlthO- and non-orthO chlorinated congeners. As a consequence, toxicological assessment is complicated by the differences in mechanisms Of toxicity, potency and other factors among congeners. The study Of individual congeners Is important not only for risk assessment but also for determination Of the mechanisms Of action. TO simplify the toxicological profile Of PCBs they have been divided in two groups: the orthO-PCBs and the non-OIthO PCBs. The on‘hO-PCBs have dioxin-like properties and are active at sub-micromolar concentrations, whereas the non-orthO-PCBs have no dioxin-like activity and require micromolar concentrations to interfere with biochemical pathways. The aim Of the present study was tO determine the molecular signaling involved in the activation Of PLAZ by the orthO-chlorinated PCB congener 2,2’,4,4’- tetrachlorobiphenyl. 101 v.3. Materials and methods V.3.A. Chemicals PCBs were purchased from ChemService (West Chester, PA). [3H][5,6,8,9,11,12,14,15]-arachidonic acid ([3H]-AA; 180—240 Ci/mmOl) was purchased from DuPont NEN (Boston, MA). W7, calmidazolium and genistein were Obtained from Sigma Chemical Company (St. Louis, MO). PD 98059, RO-32-0432, manumycin, and UO126 were purchased from Calbiochem (San Diego, CA). V.3.B. Isolation Of rat peritoneal neutrophils Neutrophils were isolated from the peritoneum Of male, Sprague-Dawley, retired breeder rats by glycogen elicitation as described (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.35, containing 1.6 mM CaClz. The percentage Of neutrophils in the cell preparations was > 95%, and the viability was >95% determined by the ability to exclude trypan blue. The isolation procedure was performed at room temperature. V.3.C. Exposure to PCBs PCB stock solutions were prepared by dissolution in N,N- dimethylformamide (DMF). Neutrophils (2x10°) were suspended in HBSS (1 mL) in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 102 v.3. Materials and methods V.3.A. Chemicals PCBs were purchased from ChemService (West Chester, PA). [3H][5,6,8,9,11,12,14,15]-arachidonic acid ([3H]-AA; 180-240 Ci/mmol) was purchased from DuPont NEN (Boston, MA). W7, calmidazolium and genistein were Obtained from Sigma Chemical Company (St. Louis, MO). PD 98059, RO-32-0432, manumycin, and UO126 were purchased from Calbiochem (San Diego, CA). V.3.B. Isolation Of rat peritoneal neutrophils Neutrophils were isolated from the peritoneum Of male, Sprague-Dawley, retired breeder rats by glycogen elicitation as described (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.35, containing 1.6 mM CaClz. The percentage Of neutrophils in the cell preparations was > 95%, and the viability was >95% determined by the ability tO exclude trypan blue. The isolation procedure was performed at room temperature. V.3.C. Exposure to PCBs PCB stock solutions were prepared by dissolution in N,N- dimethylformamide (DMF). Neutrophils (2x10°) were suspended in HBSS (1 mL) in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 102 uL Of the PCB stock solution was added to the tubes to achieve the desired concentration. Control neutrophils received 1 (IL Of DMF. V.3.D. Determination Of PLA2 activity Neutrophils (107/mL) were suspended in Mg2+- and Caz'ifree HBSS containing 0.1% bovine serum albumin and incubated in the presence Of 0.5 uCi/mL [°H]-AA for two hours, gently shaking at 37°C. Neutrophils were then washed twice with M921 and Ca2+-free HBSS. The cell count was adjusted so that the final concentration Of neutrophils was 2x10°lmL. Total cellular uptake Of [°H]-AA was measured in a 1-mL aliquot Of suspended cells: the incorporation of [3H]-AA was routinely between 80-88%. Release Of [3H]-AA from labeled neutrophils was measured in cells incubated with inhibitors for 20 minutes (37°C) and then exposed tO 25 (M 2,2’,4,4’- tetrachlorobiphenyl at 37°C for 30 min, unless otherwise stated. At the end of each incubation, neutrophils were placed on ice and spun in a centrifuge at 0°C for 10 min. The cell-free supernatant fluids were transferred tO vials containing scintillation cocktail (14 mL), and the total radioactivity in each sample was determined by liquid scintillation counting. 103 V. 3. E. Detection of phosphorylated cPLAz and p42/p44 MAPKs Neutrophils (3 x 10° /mL) were suspended in HBSS and incubated with inhibitors for 20 min at 37°C before stimulation with 25 uM 2,2’,4,4’-tetrachlorobiphenyl or vehicle and incubated for an additional 30 min at 37°C. After incubation, samples were spun in a centrifuge at 4°C for 10 min. The pellets were washed twice with phosphate-buffered saline (PBS) (pH 7.4) and resuspended in 300 uL lysing buffer (20% sodium dodecyl sulfate, SDS) for one hour and then sonicated for 30 sec. Samples were denatured by boiling for 5 min at 100°C and separated on a denaturing, 10% SDS polyacrylamide gel. Approximately 50 pg Of protein were added to each lane. Proteins were transferred electrophoretically tO nitrocellulose membranes. After transfer, membranes were blocked for 3-4 hours in Tris- buffered saline (TBS) + Tween 20 (1%) (TBS-T) containing 4% chicken ovalbumin and 0.25% sodium azide. Membranes were incubated with mouse anti-phosphorylated MAPK antibody (New England BiOLabs) (12500) or the anti-cPLAz antibody (Santa Cruz Biotechnology) (1:500) in blocker for two hours with constant rocking. Blots were washed three times with TBS-T (30, 5 and 5 min) and once with TBS (5 min). Goat anti-mouse IgG linked to horseradish peroxidase (1:7500) in TBS was added for 1 hour. Blots were washed using the same protocol as described above. Enhanced 104 chemiluminiscence using Amersham reagents was performed to visualize labeled, phosphorylated proteins. V.3.F. Cytotoxicity assay Release by neutrophils Of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium was used as an indicator Of cytotoxicity. The method for sample preparation was the same as that described above. LDH activity present in the supernatant fluid was measured according tO the method Of Bergmeyer and Bernt (1974). V.3.G. Statistical Methods Data are presented as the means : SEM from at least 4 different experiments conducted in triplicate. TO calculate percent Of control for release Of [3H]-AA in studies using inhibitors, first the values for release Of [3H]-AA in the absence Of 2,2’,4,4’-tetrachlorobiphenyl at each concentration Of inhibitor (including vehicle control) were subtracted from the corresponding values in the presence Of 2,2’,4,4’-tetrachlorobiphenyl to calculate the specific 2,2’,4,4’-tetrachlorObiphenyl-induced release. Second, the value for 2,2’,4,4’-tetrachlorobiphenyl-induced release in the absence Of inhibitor (vehicle control) was taken tO be 100%. Angular transformation (arcsin) was used on percentage data to generate an approximated gaussian distribution. Detection Of significant differences among treatments was determined using 105 analysis Of variance (ANOVA) and Tukey test as a post hoc test. When angular transformation did not produce gaussian data, Kruskal Wallace ANOVA on Ranks was used. Two-tailed p-values <0.05 were considered significant. v.4. Results V.4.A. Activation of PLA2 by 2,2’,4,4’-tetrachlorbiphenyl and 3,3’,4,4’-tetrachIorobiphenyl. Dose-response curves for the activation Of neutrophil PLAZ by 2,2’,4,4’-tetrachlorobiphenyl and 3,3’,4,4’-tetrachlorobiphenyl are presented in Figures V1 and V2, respectively. Only the orthO-chlorinated congener activated PLAZ and did so in a dose-dependent manner. At concentrations greater than 50 uM, 2,2’,4,4’-tetrachlorobiphenyl induced a statistically significant release Of LDH from neutrophils that was less than 20% Of total activity. BEL (25 pM) decreased z 50% of the total PLAZ activity induced by 2,2’,4,4’-tetrachlorobiphenyl, suggesting that this congener targets the Ca2+-Independent PLA2 isoform (iPLAz). 106 Percentage Of Activity 120 * * O PLA2 100 - . * l:l LDH 80 - 60 - 40 - 20 . * * * * o .. 0.0 0.01 0.1 1 10 100 2,2',4,4'-TetrachlorObiphenyl (pM) Figure v.1. Effects of 2,2’,4,4’-tetrachlorobiphenyl on neutrophil PLA2 activity and cytotoxicity. Neutrophils were labeled with [3H]-AA and incubated with the concentrations shown, as stated in Materials and methods. For this and all subsequent graphs, the activity is plotted against the log Of the concentration. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. Aroclor 1242 under these conditions caused release Of 25-35% Of total incorporated [3H]-AA. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 107 Percentage Of Activity 100 80 _ O PLA2 l LDH 60 - 40 a 20 - I r 4* J 0 - O— 1 O I 3:: 0 0.01 0.1 1 10 25 3,3',4,4'-TetrachIorobiphenyl (pM) Figure v.2. Effects of 3,3’,4,4’-tetrachlorobiphenyl on neutrophil PLA2 activity and cytotoxicity. Neutrophils were labeled with [3H]-AA and incubated with the concentrations shown, as stated in Materials and methods. The release of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. LDH released at each concentration was compared with the LDH released from neutrophils lysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. 108 V.4.B. Role Of protein kinase pathways and calmodulin in 2,2’,4,4’-tetrachIorobiphenyl-induced activation Of PLAz TO study different signaling cascades that could be involved in the activation Of PLA2 induced by 2,2’,4,4’-tetrachlorobiphenyl, various protein inhibitors and antagonists were tested. The tyrosine kinase (TK) inhibitor, genistein, inhibited a small fraction Of the 2,2’,4,4’-tetrachlorobiphenyl-induced PLA2 activity in a dose- dependent manner (Figure V.3). Maximal inhibition (s20%) was reached at 50-100 pM. PD 98059 and UO126, two inhibitors Of the mitogen activated protein kinase (MAPK) kinase (MEK) produced a decrease in 2,2’,4,4’-tetrachlorobiphenyl-stimulated PLAZ activity which was only significant for UO173 and reached approximately 20% inhibition (Figure V.4). RO-32-0432 (10 pM), an inhibitor Of protein kinase C (PKC), produced an inhibition Of stimulated PLA2 activity that was similar In magnitude tO that Observed for genistein and UOI73 (Figure V.5). Finally, manumycin, an inhibitor Of ras farnesylation, produced a greater degree Of inhibition (approximately 35%) at 10 (M (Figure v.6). These inhibitors were not cytotoxic (< 25% LDH release) at the concentrations tested. The effects Of two chemically distinct calmodulin inhibitors were tested. At noncytotoxic concentrations, W7 and calmidazolium, each 109 120 a 3100- - a I T ,;<_ i I O 0: 80+ 3 * C d) 2 604 * o D. 40 . . . . - - 0 0.01 0.1 1 10 100 Genistein (pM) Figure V.3. Dose-response curve for inhibition Of 2,2’,4,4’- tetrachlorobiphenyl (25 nM)-induced release of [3H]-AA by genistein. Cells were incubated with the indicated concentrations Of genistein as described in Materials and methods. Data are expressed as means : SEM of four different experiments with triplicates per assay. *, p<0.05 vs. [3H]-AA released by neutrophils exposed tO 25 pM 2,2’,4,4’- tetrachlorobiphenyl alone. 110 Percentage Of activity 110 ' i 100 - 0 90 - V - PD 93059 g 80 - - UO126 70 - 60 . . . . . . 0 0.01 0.1 1 10 100 Drug (pM) Figure V.4. Dose-response curves for inhibition of 2,2’,4,4’- tetrachlorobiphenyl (25 nM)-induced release of [3H]-AA by PD 98059 and UO126. Cells were incubated with the indicated concentrations Of PD 98059 and UO126 as described in Materials and methods. Data are expressed as means i SEM Of four different experiments with triplicates per assay. *, p<0.05 vs. [3H]-AA released by neutrophils exposed to 25 pM 2,2’,4,4’- tetrachlorobiphenyl alone. 111 120 a 5100 - 2 .- “5 I i o 80 - * CI :3 C d) 2 60 - d) D. 40 I l I I 0.0 0.01 0.1 1.0 10.0 RO-32-0432 (pM) Figure V.5. Dose-response curve for inhibition of 2,2’,4,4’- tetrachlorobiphenyl (25 nM)-induced release Of [°H]-AA by Ro-32-0432. Cells were incubated with the indicated concentrations Of RO-32-0432 as described in Materials and methods. Data are expressed as means i SEM Of four different experiments with triplicates per assay. *, p<0.05 vs. [°H]-AA released by neutrophils exposed to 25 pM 2,2’,4,4’- tetrachlorobiphenyl alone. 112 160 .2 3120- $100- 5') s 5 80- 2 * d) D. 60. 40 1 . 0.0 0.01 0.1 1.0 10.0 Manumycin A (pM) Figure V.6. Dose-response curve for inhibition of 2,2’,4,4’- tetrachlorobiphenyl (25 pM)-induced release of [3H]-AA by manumycin A. Cells were incubated with the indicated concentrations Of manumycin A as described in Materials and methods. Data are expressed as means : SEM Of four different experiments with triplicates per assay. *, p<0.05 vs. [3H]-AA released by neutrophils exposed to 25 pM 2,2’,4,4’- tetrachlorobiphenyl alone. 113 induced a significant decrease (s40%) in the 2,2’,4,4’-tetrachlorobiphenyl- induced PLA2 activity (Figure V.7). V.4.C. Effects Of 2,2’,4,4’-tetrachlorObiphenyl and 3,3’,4,4’-tetrachIorobiphenyl on cPLAz and p42/p44 MAPK- phosphorylation. 2,2’,4,4’-tetrachlorobiphenyl induced the phosphorylation Of cPLA2 in rat neutrophils. This was not Observed with the non-ortho- congener 3,3’,4,4’-tetrachlorobiphenyl (Figure V.8). Similarly, 2,2’,4,4’- tetrachlorobiphenyl induced the phosphorylation Of p44 MAPK, commonly referred to as extracellular signal-regulated kinase (ERK-1), and increased the phosphorylation Of the constitutively expressed, phosphorylated p42 MAPK (ERK-2) (Figure V.9). Induction Of phosphorylation Of p44 MAPK was blocked by PD 98059 and attenuated by genistein. Although western analysis does not measure activity, increased phosphorylation Of p42/p44 MAPK is in general accompanied by increased activity (Hashimoto et al., 1999; Qiu and Leslie, 1994) so that these results imply that 2,2’,4,4’-tetrachlorobiphenyl increased activity Of cPLA2 and p44 MAPK. 114 120 _ Control I— : W7 20 pM 1 @100 _ W7 40 pm .2 CE: Calmidazolium 1 pM ‘ 1H 0 <6 80 - * 2": “5 * a: 60 - O) .S c u a, 40 2 a: 20 - m o ' I I Drug (pM) Figure V.7. Effects of calmodulin inhibitors on 2,2’,4,4’- tetrachlorobiphenyI-induced PLA; activity. Cells were incubated with the indicated concentrations of calmodulin inhibitors W7 or calmidazolium as described in Materials and methods. Data are expressed as means i SEM Of four different experiments with triplicates per assay. *, p<0.05 vs. [°H]-AA released by neutrophils exposed to 25 pM 2,2’,4,4’- tetrachlorobiphenyl alone. 115 B‘scPLA; _.__,. CPLA; my . l f DMF 3:3,,404” 2,2,14,4’- TCB TCB Figure v.8. 2,2’,4,4’-tetrachIorobiphenyl but not 3,3’,4,4’- tetrachlorobiphenyl phosphorylates cPLAz. 3x106 cells/mL were incubated with 1 pL vehicle control (DMF) or with 25 pM of each PCB congener for 30 min at 37 °C. cPLAz phosphorylation was detected by Western blotting as described in Materials and methods. Western blot is representative Of two independent experiments. 116 44___> 342—» c-Iw .- DMF 3,334,42 2,234,4'- Genistein PD 98059 rca TCB 50 um 50 um 25 um 25 uM + . 2,2’,4,4-rc13 25 pM Figure V.6. p42/p44 MAPK phosphorylation response by PCBs: Effects of genistein and PD 98059 in 2,2'4,4’-tetrachlorobiphenyI-induced phosphorylation of p42/p44 MAPK. 3x10° cells/mL were incubated with 1 pL vehicle control (DMF) or with 25 pM of each PCB congener for 30 min at 37 °C. Experiments with inhibitors were conducted by incubating the cells with the shown inhibitor concentration for 20 min (37 °C) and then with 2,2’,4,4-tetrachlorobiphenyl for additional 30 min. p42/p44 MAPK phosphorylation was detected by Western blotting as described in Materials and methods. Western blot is representative for two independent experiments. 117 v.5. Discussion It has been demonstrated that the PCB mixture Aroclor 1242 activates PLA2. Results reported here show that 2,2’,4,4’- tetrachlorobiphenyl but not 3,3’,4,4’-tetrachlorobiphenyl activates PLAZ, suggesting that it is the orthO-chlorinated congeners in Aroclor 1242 which are responsible for the activity. Furthermore, results suggest that 2,2’,4,4’- tetrachlorobiphenyl activates two isoforms Of PLA2: a BEL-sensitive PLA2 known to be Cay-independent (iPLAz) and CPLAz. In this study, the effects Of different signaling pathways leading tO activation Of PLA2 by 2,2’,4,4’-tetrachlorobiphenyl were studied using pharmacological inhibitors. It was shown that TKs, PKC, ras and MEK, as well as calmodulin are important for a fraction Of the Observed PLA2 activity induced by this PCB congener. Among PLAzs, only cPLAz has been shown tO be regulated by phosphorylation (Lin et al., 1998; Qiu and Leslie, 1994; Borsch-Haubold et al., 1998). Due to the fact that activation of cPLAz may occur following a signal transduction pathway involving TK, PKC, ras and MEK (Lin et al., 1998; Miura et al., 1999; Sa and Das, 1999; Li et al., 1997), it is likely that inhibition Of these pathways prevents activation Of this isoform Of PLA2. Consequently, it is possible that the fraction Of the Observed PLA2 activity inhibited by interference with these pathways represents the contribution Of cPLAZ. 118 Previous studies have shown that PCBs can induce tyrosine phosphorylation in rat neutrophils (Tithof et al., 1997). In this study, genistein (50-100 uM), a nonspecific tyrosine kinase inhibitor, abrogated the PLAZ response elicited by 2,2’,4,4’—tetrachlorobiphenyl. Different genistein concentrations have been used to block some effects induced by tyrosine kinase activation, such as prostaglandin production (5-10 uM) (Kniss et al., 1996), adherence (30-200 pM) (Gozin et al., 1998; Ozaki et al., 1993), superoxide anion release (50-300 uM) (Hazan et al., 1997), and degranulation (5-200 pM) (Foster et al., 1999). Inhibition Of ras attenuated activation Of PLA2 by 2,2’,4,4’- tetrachlorobiphenyl. Ras is a GTP-binding protein that regulates a variety Of cellular processes such as cell growth and differentiation (SatOh et al., 1992). Ras protein acts as a signal transducer in which signaling from tyrosine kinases, Intrinsic tO or associated with receptors, activates ras in response to an extracellular stimulus. Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. Ras activity requires its attachment tO the inner surface Of the plasma membrane, which is Obtained by the addition Of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (Rowinsky et al., 1999). Manumycin is an antibiotic with recognized activity tO inhibit protein farnesyltransferase (Hara et al., 1993). It has been used tO block p21 (ras)-dependent pathways (Lee-Kwon 119 et al., 1998; Flamigni et al., 1999; Smith et al., 1999) such as phosphorylation Of p42/p44 MAPK (Kouchi et al., 1999). In vitrO studies have shown that manumycin can block human protein farnesyltransferase activity with an IC50 Of 1.8 pM (Del Villar et al., 1999). Inhibitors Of farnesyltransferases have emerged as potential anti-cancer drugs (Sattler and Tamonoi, 1996; Waddick and Uckun, 1998), based on the involvement Of ras in cell growth and morphogenesis (Gibbs and Oliff, 1997). The activation of a ras-dependent pathway can be a link between PCBs and tumor formation, particularly because the PCB mixture Aroclor 1254 (Borlak et al., 1996) and the on‘hO-chlorinated PCB 2,3,4,2’,4’,5'- hexachlorobiphenyl (Gribaldo et al., 1998) have been shown to induce overexpression Of ras. The PKC inhibitor RO-32-0432 partially inhibited PLA2 activity induced by 2,2’,4,4’-tetrachloroblphenyl. RO 32-0432 is a bisindolylmaleimide molecule that selectively inhibits PKCO, or PKCB with low selectivity for PKC, (Wilkinson et al., 1993). The involvement Of PKC in PLAZ activation by PCBs is not surprising because it is known that PCBs can interact directly with PKC and promote its translocation (Kodavanti et al., 1998). 2,2’,4,4’-Tetrachlorobiphenyl phosphorylated p44 MAPK and increased the phosphorylation Of p42 MAPK. This effect on p44 MAPK was partially blocked by genistein and abrogated by PD 98059. In rat 120 neutrophils, activation Of MAPK induced by fMLP is sensitive in a dose- dependent manner tO inhibitors Of TK, PKC, PLC and chelators Of intracellular Ca2+ (Chang et al., 1999), suggesting that MAPKs are a converged downstream target for extracellular signaling by fMLP. p42/p44 MAPKs can be phosphorylated by MAPK kinase (MEK), an enzyme inhibited by PD 98059 (Dudley et al., 1995). This compound prevents the activation Of MAPK kinase 1 by Raf or MEK kinase in vitrO with an IC50 Of 2-7 pM. However, PD 98059 inhibited the activation Of MAPK kinase 2 by Raf at a larger IC50 value (50 pM) (Alessi et al., 1995). Another inhibitor Of the MEK pathway used in this study was UO126. This compound selectively inhibits MEK-1 and MEK-2 (Favata et al., 1998) and had similar effects on PLA2 activity as PD 98059. Taken together, the Observed phosphorylation Of cPLA2 and the effects Of the protein inhibitors in the 2,2’,4,4’-tetrachlorObiphenyl-induced PLA2 activity suggest that phosphorylation/activation Of this enzyme occurs following the signal transduction cascade: PKC or TK::>ras (Raf)2>MEK:>MAPK:>cPLA2, as a typical response tO cellular stress or mitogenic signals (Norris and Baldwin., 1999; Takahashi et al., 1999; Qiu and Leslie, 1994). In contrast tO cPLAz, the Ca2+-independent PLA2 (iPLAz) is not regulated by phosphorylation but can be modulated by protein-protein interaction with calmodulin (Wolf and Gross, 1996). As Observed with 121 Aroclor 1242 (Chapter IV), non cytotoxic concentrations Of the calmodulin inhibitors calmidazolium and W7 were slightly more effective than the kinase inhibitors tO inhibit 2,2’,4,4’-tetrachlorObiphenyI-induced activation Of PLA2. In bovine tracheal smooth muscle strips W7 and calmidazolium inhibit the CaZ+—calmodulin-induced activation Of myosin light chain kinase with IC50 values Of 25 pM and 1 pM, respectively (Asano, 1989), which closely match the effective inhibitory concentrations used in this study. Molecular structure Of the calmodulin complexed with W7 has been reported (Osawa et al., 1998). W7 binds to hydrophobic amino acid residues which occur in the vicinity Of Ca2+ binding sites and elicits structural changes similar to trifluoperazine and oxmetidine (Akiyama and SutOO, 1988). Upon binding, W7 tends tO form a globular structure in solution (Osawa et al., 1999). Studies Of calmodulin with W7 have suggested that this protein can accommodate a variety Of bulky, aromatic rings into the two hydrophobic pockets (Osawa et al., 1998), supporting a hypothesis that PCBs may be ligands for this Ca2+-regulatory protein. Many molecules can bind calmodulin, for instance tamoxifen (McCague et al., 1994), the antitumor drugs KAR-2 and vinblastlne (Vertessy et al., 1997; Orosz et al., 1997), and cyclosporin-A (Knott et al., 1994) among others. It has been recently demonstrated that calmodulin can bind simultaneously different drugs such as trifluoperazine and KAR-2 0r vinblastlne and KAR-2 (Vertessy et al., 1998). An interesting possibility 122 is that calmodulin can be a carrier protein for PCBs, allowing PCBs to interact directly with the iPLA2 while the calmodulin-iPLA2 complex is formed through a different binding site. Experimental evidence showing the binding Of PCBs to calmodulin will be necessary tO prove some Of the effects Observed with calmodulin inhibitors. However, it is likely that PCBs are binding to calmodulin because Of their hydrophobic nature and presence Of aromatic rings, two structural requirements for drug binding to this protein (Hait et al., 1986). In short, the orthO-PCB 2,2’,4,4’-tetrach|orobiphenyl, but not the non-on‘hO-PCB 3,3’,4,4’-tetrachlorObiphenyl, activated neutrophil PLA2. This PLA2 activity was sensitive to BEL, suggesting the stimulation Of iPLA2. Experiments with calmodulin inhibitors indicate that calmodulin can be an important regulator Of the iPLAz activity induced by orthO-PCBs. Phosphorylation Of cPLAz and p42/p44 MAPK and inhibition Of a fraction Of the Observed PLAZ activity by different kinase inhibitors presumes that in addition tO iPLA2, cPLA2 is also activated by 2,2’,4,4’-tetrachlorobiphenyl through a mechanism involving TK, PKC, ras, and the MEK pathway. 123 Chapter VI STRUCTURE-ACTIVITY RELATIONSHIPS FOR THE ACTIVATION OF RAT NEUTROPHIL PHOSPHOLIPASE A2 BY ORGANOCHLORINE COMPOUNDS 124 Vl.1. Summary Organochlorine compounds (OCs) are some Of the main toxicants present in the fOOd web and target different cellular systems including the non-specific immune system. The Objective Of this study was tO test the hypothesis that OCs that activate neutrophils share common structural features. Using activation Of phospholipase A2 (PLA2) as a marker Of neutrophil activation, isolated rat neutrophils were exposed tO a variety Of OCs. The on‘hO-substituted polychlorinated biphenyl 2,2’,4,4’- tetrachlorobiphenyl, the Ot-, 8- and y-hexachlorocyclohexanes (HCCHs), p,p’-diorthOdichlorotrichloroethane (DDT), dieldrin and chlordane, but not the non-ortho substituted 3,3’,4,4’-tetrachlorobiphenyl or B-HCCH induced activation Of PLA2 in neutrophils. This activation Of PLA2 was sensitive to bromoenolactone (BEL), suggesting that a Cay-independent isoform Of PLA2 is activated by these compounds. Molecular modeling techniques were used to develop structure-activity relationships for the activation Of PLA2 by OC compounds. Superimposing three-dimensional structures we have identified an electrotopological motif shared by all the active compounds, including the iPLAz inhibitor BEL. This motif is absent in the inactive B-HCCH and non-ortho chlorinated PCBs. These results suggest that the bioactivity Of organochlorine compounds in neutrophils may be due tO the presence Of a specific substructure that fits into a receptor-like structure, allowing the activation Of the neutrophil PLA2. This motif, which 125 we have called the OG motif, consists Of a planar hydrophobic domain connected rigidly at a perpendicular angle to a negatively charged atom. VI.2. Introduction One Of the forces that moved the industrial age was the extensive use Of new chemicals which allowed the production Of large quantities Of food supplies and the decrease in child mortality. Among these chemicals, organochlorine (OC) compounds were Of particular importance due to their versatility and low cost. These same chlorinated chemicals such as polychlorinated biphenyls and chlorinated pesticides such as DDT, dieldrin, hexachlorocyclohexanes and chlordane are now recognized as fOOd contaminants due to their accumulation in the food chain. Adding tO concern about OCs is their diverse toxicity. The immune system, and particularly the neutrophil, has been shown to be a target for OC compounds such as PCBs (Ganey et al, 1993), hexachlorocyclohexanes (HCCHs) (Dunstan et al., 1996), DDT (Sitarska et al., 1990), dieldrin (Hewett and Roth, 1988) and chlordane (Miyagi et al., 1998), among others. For example, PCBs activate neutrophils through complex biochemical mechanisms tO induce superoxide anion release and degranulation. These processes are mediated through the activation Of PLA2. 126 Toxicological testing Of all the known OC fOOd contaminants for their activity on neutrophil function is not practical because it is expensive and time consuming. A knowledge Of the molecular structure and the biological activity Of groups Of related compounds will allow the development Of structure-activity relationships (SARs) which can be critical not only tO predict the activity Of unknown, related compounds but also for risk assessment. Different SAR studies have been performed for PCBs (Van der Burght et al., 1999; Brown et al., 1998; Kodavanti et al., 1997; Mekenyan et al., 1996), cyclodienes (ROhr et al., 1985), DDT (Coats, 1990), and other halogenated pesticides (Moser et al., 1993). In neutrophils, SAR studies have been conducted for the activity Of PCBs to induce superoxide anion production (Brown et al., 1998) and degranulation (Olivero and Ganey, 1998). This work presents the results Of an SAR study conducted to find molecular features responsible for the activation Of PLA2 by OC compounds. VI. 3. Materials and methods VI. 3. A. Chemicals [3H][5,6,8,9,11,12,14,15]-AA ([3H]-AA; 180-240 Ci/mmol) was purchased from DuPont NEN (Boston, MA), BEL was purchased from Biomol (Plymouth Meeting, PA). Organochlorine compounds 2,2’,4,4’- tetrachlorobiphenyl, 3,3’,4,4’-tetrachlorobiphenyl, 0t-, B-, 8-, and y- 127 hexachlorocyclohexane, 1 ,2,3,4,1 0,10-hexachlorO-6,7-epoxy- 1,4,4a,5,6,7,8,8a-octahydrO-1,4,5,8-dimethanonaphthalene (dieldrin), p,p’- dichlorOdiphenyltrichloroethane (DDT) and 1,2,4,5,6,7,8,8-Octachlor- 2,3,3A,4,7,7A-hexahydrO-4,7-methanolndane (chlordane) were purchased from ChemService (West Chester, PA). VI. 3. B. Isolation of rat peritoneal neutrophils Neutrophils were isolated from the peritoneum Of male, Sprague-Dawley, retired breeder rats by glycogen elicitation (Hewett and Roth, 1988). Isolated neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.35, containing 1.6 mM CaClz. The percentage Of neutrophils in the cell preparations was > 95%, and the viability was >95% determined by the ability tO exclude trypan blue. The isolation procedure was performed at room temperature. VI. 3. C. Exposure tO organochlorine compounds Organochlorine (OC) stock solutions were prepared by dissolution in N,N-dimethylformamide (DMF). Neutrophils (2x106) were suspended in HBSS (1 mL) in borosilicate glass test tubes, 12 x 75 mm (VWR, Chicago, IL), and 1 pL Of the OC stock solution was added to the tubes to achieve the desired concentration. Control neutrophils received 1 uL Of DMF. 128 VI. 3. D. Determination of PLA2 activity Neutrophils (107/mL) were suspended in Mgz‘i and Ca2+-free HBSS containing 0.1% bovine serum albumin and incubated in the presence Of 0.5 pCi/mL [3H]-AA for two hours, gently shaking at 37°C. Neutrophils were then washed twice with Mg”- and Ca2+-free HBSS. The cell count was adjusted so that the final concentration Of neutrophils was 2x10°/mL. Total cellular uptake Of [3H]-AA was measured in a 1-mL aliquot of suspended cells: the incorporation Of [°H]-AA was routinely between 80-88%. Release Of [3H]-AA from labeled neutrophils was measured in cells treated with OCs for 30 minutes (37 °C). Studies with the iPLAz inhibitor BEL were conducted by incubating the cells with BEL for 20 mins (37 °C) and then with OC compounds for an additional 30 minutes at the same temperature. At the end Of each incubation, neutrophils were placed on ice and spun in a centrifuge at 0°C for 10 min. The cell-free supernatant fluids were transferred tO vials containing scintillation cocktail (14 mL), and the total radioactivity in each sample was determined by liquid scintillation counting. IV. 3. E. Cytotoxicity assay Release by neutrophils Of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium was used as an indicator Of cytotoxicity. LDH activity present in 10 pL Of the supernatant fluid from the 129 PLA2 assay was measured according to the method Of Bergmeyer and Bernt (1974). VI. 3. F. Structure-activity relationships VI.3.F.i Data set Organochlorine compounds tested for their ability to activate PLA2 were 2,2’,4,4’-tetrachlorobiphenyl, 3,3’,4,4’- tetrachlorobiphenyl, 0t-, B-, 8-, and y-hexachIorocychlohexane, DDT, dieldrin and chlordane. Compounds that induce a statistically significant release Of arachidonic acid at 25 pM when compared to that elicited by the vehicle control (DMF) were considered active. Molecular structures for the compounds in the data set appear in Figure Vl.1. VI.3.F.ii. Computational details. Molecular modelling procedures used in this study were performed using Hyperchem 5.1 (Hypercube Inc, 1996). The compounds in the data set were entered as two-dimensional sketches into Hyperchem and stored as atomic coordinates. The presence Of a torsional angle in the PCB structures and DDT generates different conformers, among which some are less energetically favorable. TO search for the most stable structure, molecule geometries for these compounds were submitted to a conformational search tO Obtain aconformer with the lowest energy (ie, 130 DDT Dieldrin Chlordane Figure VI.I. Molecular structure Of organochlorine compounds used in this study. 131 the most stable conformer). Full optimization geometry for the best conformer for PCBs, DDT, and the other OC compounds was performed using the semiempirical method AM1 (Dewar, 1985) running on Hyperchem. Electronic properties were calculated from single point calculations at the ab initiO level STO-3G (Hehre et al., 1969). Other properties from the energetically minimized structure, such as the molecular weight and the total solvent-accessible surface area were calculated using the subroutine QSAR properties implemented in ChemPlus/ Hyperchem 5.1. VI.3.F.iii. Model construction Establishment Of relationships between the molecular structure and the PLA2 activity for the OC compounds in the data set was done following a structure-based design approach. The intrinsic biological/toxicological activity or potency can be a function Of the three- dimensional shape Of the molecule and Of the electric charge distribution and lipophilicity. These are also determinant factors in the molecular affinity for a receptor. Accordingly, comparison Of conformational profiles of various molecules tO a template may reveal those overlapping or similar conformational spaces and electronic features which are common to the molecules that elicit a particular effect. General similarities between the OC were searched by superposition Of selected atoms Of an OC compound on their chemically equivalent atoms on the 2,2’,4,4’- 132 tetrachlorobiphenyl used as a template. The Hyperchem subroutine ChemPlus was used tO perform the calculations. Initially, the file for the optimized 2,2,’4,4’-tetrachlorobiphenyl molecule was open and merged with the targeted optimized geometry. For each molecule six atoms were selected, and the function “RMS overlay” was used to perform the superpositions. The program translates the centers Of the atoms tO be fitted to the centroid Of the corresponding atom in the reference molecule. The best fit was Obtained when the structure superposition gave the lowest root-mean-square deviation (RMSD) value between selected pairs Of equivalent atoms using the 2,2’,4,4’-tetrachlorobiphenyl as a reference. After visual comparison Of the superpositions Of 2,2’,4,4’- tetrachlorobiphenyl with the active vs. the inactive OCs, the molecular features, both topological and electronical, present or absent in both groups were determined (SAR model). In order to validate the model, non- cytotoxic doses Of different organochlorine and organobromine compounds were tested tO see if they activated PLA2 following the structural rules presented by the SAR model. The general procedure followed to Obtain the SAR is presented in Figure VI.2. 133 Data set l—— l Determination Of Molecular modeling PLAz activity l l H Search for best conformers . . St ct re t' ' t' Inactive Active m " Op 'm'za '°" Calculation of properties v —> Molecule superposition p onto template l Identification Of molecular features present in the active but absent in the inactive compounds SAR model I External validation Of the model Figure VI.2. General scheme for the development Of structure- activity relationships for organochlorine compounds and activation or rat PLAz. 134 VI. 4. Results VI.4.1. PLAz activity induced by organochlorine compounds. The dose-response curve for the activation Of rat neutrophil PLAz by different OC compounds showed clear differences in activity. 2,2’,4,4’-tetrach|orobiphenyl activated PLA2 at concentrations 2 5 pM. Its activity at concentrations 2 10 (M was similar tO that elicited by 10 pg/mL Aroclor 1242 (See Chapter V, Figure V.1). Concentrations greater than 50 uM produced some cytotoxicity (s20% LDH release) (Figure V.1). 3,3’,4,4’-tetrachlorobiphenyl failed tO activate PLA2 at concentrations up tO 25 pM (Figure v.2). Greater concentrations were not compatible with the buffer and precipitated. a-, 53-, and y-HCCH activated PLA2 at concentrations equal to or greater than 10 pM (Figures VI.3, VI.4, and VI.5). Of these hexachlorocyclohexanes, a-HCCH was the least efficacious. NO cytotoxicity was Observed for any Of these OCs at the maximal concentrations tested (100 pM). Conversely, B-HCCH (Figure Vl.6) was unable to activate PLAz over the concentration range tested (0- 100pM) DDT, dieldrin and chlordane each activated PLA2 at concentrations equal to or greater than 10 pM (Figures Vl.7, V|.8 and Vl.9). Only dieldrin 135 100 O PLA2 5s 80- El LDH E ‘6 E 60- * O o m 40‘ B 5 B 20- * g * 0' * 0 0.01 0.1 1 10 100 Alpha Hexachlorocyclohexane (pM) Figure VI.3. Effects Of a-HCCH on neutrophil PLA2 activity and cytotoxicity. Neutrophils were labeled with [3H]-AA and incubated with the concentrations shown, as stated in Materials and methods. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 ug/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. Under these conditions, Aroclor 1242 released 25-35% Of total [3H]-AA. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 136 Percentage Of Activity 100 . PLAZ * 80' LDH I 60 1 40- 20 - * ' :7 f: I O . 0 0.31 0:1 17 170 100 Delta-HexachIorocyclohexane (pM) Figure V , and cytotoxicity. Neutrophils were labeled with [3H]-AA and incubated with the concentrations shown, as stated in Materials and methods. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means : SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 137 100 3. 80- 25 1'5 < 60~ u... O o C) 40— .9 c o B 20- o (L 0a 0 0:01 0? 1 1'0 100 Gamma Hexachlorocyclohexane (pM) Figure VI.5. Effects Of rHCCH on neutrophil PLA2 activity and cytotoxicity. Neutrophils were labeled with [3Hj-AA and incubated with the concentrations shown, as stated in Materials and methods. The release of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 138 Percentage Of Activity 100 O PLA2 80- LDH 60 " 40 ' 20 ' o. ‘ him 0 0:01 0:1 1' 10 1 00 Beta Hexachlorocyclohexane (pM) Figure Vl.6. Effects of )B-HCCH on neutrophil FLA; activity and cytotoxicity. Neutrophils were labeled with [°H]-AA and incubated with the concentrations shown, as stated in Materials and methods. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage of activity Of Aroclor 1242. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 139 160 140: 120 - 100 - Percentage Of Activity N h 0? on O O O O O I l I l I IL) 6 0.0 0.01 0.1 1:) 10.0 100.0 DDT (pM) Figure VI.7. Effects Of DDT on neutrophil phospholipase A; activity and cytotoxicity. Neutrophils were labeled with [3H]-AA and incubated with the concentrations shown, as stated in Materials and methods. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 140 200 O 3‘ n E 150 4.: o < ‘6 o 100 - U: I! t: a: e 50- cu D. o - ‘.. 0.0 0.01 0.1 1.0 10.0 100.0 Dieldrin (pM) Figure Vl.8. Effect of Dieldrin on neutrophil PLAz activity and cytotoxicity. Neutrophils were labeled with [3H]-AA and incubated with the concentrations shown, as stated in Materials and methods. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 ug/mL Aroclor 1242-treated neutrophils under the same experimental conditions and are presented as percentage Of activity Of Aroclor 1242. LDH released at each concentration was compared with the LDH released from neutrophils Iysed with 0.01% Triton X-100 and are presented as percentage Of total LDH activity. Results are expressed as means i SEM for four different experiments performed in triplicate. *. Significantly different from vehicle control (DMF). 141 Percentage Of Activity 350 . PLA2 300 - 250 - A A N O 0'! O O O O l I I 50-1 l q ' I I 0.0 0.01 0.1 1 10 100 Chlordane (pM) Figure VI.9. Effects of Chlordane on neutrophil phospholipase A; activity and cytotoxicity. Neutrophils were labeled with [°H]-AA and Incubated with the concentrations shown, as stated in Materials and methods. The release Of [3H]-AA into the medium at each concentration was compared with the release from 10 pg/mL Aroclor 1242- treated neutrophils under the same experimental conditions, and are presented as percentage Of activity Of Aroclor 1242. LDH release at each concentration was compared with the LDH release from 0.01% Triton X-100-Iysed neutrophils and are presented as percentage Of activity Of Triton X-100. Results are expressed as means i SEM from four different experiments performed in triplicates. *. Significantly different from vehicle control (DMF). 142 presented a significant cytotoxicity (>20%). This occurred at concentrations greater than 50 pM. PLA2 activity elicited by the active compounds was abrogated more than 50% by BEL (Figure Vl.10), suggesting that a CaZ+-independent PLA2 is targeted by these OCs. BEL treatment did not induce significant toxicity for any Of the tested compounds. These results are consistent with previous reports for PCBs. VI.4.2. Structure-activity relationships Molecular structures for the OCs used in this study are diverse, and apparently they have few particular similarities. Calculation Of selected electronic and topological properties Of these compounds is shown in Table Vl.1. It is evident that global properties such as the energy of the frontier orbitals highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) and dipolar moment vary considerably among compounds. Interestingly, the inactive compounds 3,3’,4,4’-tetrachlorobiphenyl and B-HCCH have the lowest dipolar moment values. In order tO lOOk for common features responsible for the activity Of 00 compounds on PLA2, the structure Of the on‘hO-chlorinated PCB 143 - Control 2,2,4,4 :100 - - Ot-HCCH e m 8-HCCH ‘E' - y-HCCH O 80 - m Dieldrin O - DDT “5 [:1 Chlordane e\° 60 - _T_ V '5 z: 40 - O (U N j 20 - D. 0 Figure Vl.10. Effects of BEL on PLA2 activity induced by CC compounds. Neutrophils were labeled with [3H]-AA and incubated with 25 pM BEL for 20 min at 37 °C followed by incubation with the OC for 30 min at 37 °C. A. The release Of [°H]-AA into the medium at each concentration was compared with the release by the same compound in absence of BEL. Results are expressed as means i SEM from four different experiments performed in triplicate. 144 Table Vl.1. Molecular descriptors for 008 in the data set. Molecules Molecular descriptors" MW SA DM MPC MNC HOMO LUMO (g/mOI) (A2) (Debye) (eV) (eV) 2,2’,4,'4-TCB** 291.99 395.6 2.573 0.098 -0.108 -8.053 5.645 3,3’,4,’4-TCB 291.99 418.9 1.273 0.088 -0.095 -7.430 4.608 a-HCCH 290.83 396.4 2.707 0.124 -0.134 -10.943 7.103 fi-HCCH 290.83 411.4 0.000 0.114 -0.116 -10.990 7.012 5-HCCH 290.83 402.2 2.788 0.125 -0.126 -10.966 7.150 y—HCCH 290.83 388.3 3.782 0.127 -0.146 -10.656 6.781 DDT 354.49 460.4 1.490 0.118 -0.117 ~7.757 5.578 Dieldrin 380.91 370.5 2.953 0.095 -0.215 -8.733 5.302 Chlordane 409.78 445.20 2.934 0.112 -0.145 -9.088 5.048 *. MW: Molecular weight; SA: solvent accessible surface area; DM: dipolar moment; MPC and MNC: Most positive and most negative charge in the molecule, respectively; HOMO: energy Of the highest occupied molecular orbital. LUMO: Energy Of the lowest unoccupied molecular orbital. **. TCB, tetrachlorobiphenyl 145 2,2’,4,4’-tetrachlorobiphenyl, an activator Of PLA2 (See chapter V), was used as a template to superimpose the structures Of the other OCs. Superposition Of a-HCCH and DDT on 2,2’,4,4’-tetrachlorobiphenyl is shown in Figure VI.11. Clearly, a—HCCH can fit on one phenyl group Of the PCB and position a chlorine in close proximity tO the orthO chlorine on the other phenyl ring. Interestingly, DDT and 2,2’,4,4’-tetrachlorobiphenyl are superimposed almost perfectly. Both superpositions showed a planar structure connected tO a chlorine perpendicular tO the plane. Superposition Of dieldrin and chlordane on 2,2’,4,4’-tetrachlorobiphenyl is presented in Figure Vl.12. Although dieldrin and chlordane lack phenyl rings, they each have a planar-like domain which fits onto one phenyl group Of the PCB, and they also can position a chlorine atom in the vicinity Of the orthO-chlorine Of the other phenyl group on the PCB. Superposition Of the inactive compounds 3,3’,4,4’- tetrachlorobiphenyl and B-HCCH on 2,2’,4,4’-tetrachlorobiphenyl is shown in Figure VI.13. Although both compounds can be superimposed to 2,2’,4,4’-tetrachlorobiphenyl through their planar motifs, they failed to match a chlorine with the chlorine in the orth- position Of the template PCB. Finally, BEL, a known inhibitor Of the Ca2+-independent PLAZ, could also be almost perfectly superimposed onto 2,2’,4,4’-tetrachlorobiphenyl (Figure VI.14). In summary, despite differences in structure, all the active 146 Figure Vl.11. Molecular structures of superimposed organochlorine compounds. A. 2,2’,4,4’-Tetrachlorobiphenyl is superimposed on or- HCCH. B. 2,2’,4,4’—Tetrachlorobiphenyl is superimposed on DDT. White lines indicate the substructure shared by both compounds. 147 Figure Vl.12. Molecular structures of superimposed organochlorine compounds. A. 2,2’,4,4’-Tetrachlorobiphenyl is superimposed on dieldrin B. 2,2’,4,4’-Tetrachlorobiphenyl is superimposed on chlordane. White lines indicate the substructure shared by both compounds. 148 Figure Vl.13. Molecular structures Of superimposed organochlorine compounds. A. 2,2’,4,4'-Tetrachlorobiphenyl is superimposed tO 3,3’,4,4’-tetrachlorobiphenyl. B. 2,2',4,4’- Tetrachlorobiphenyl is superimposed to B-HCCH. White lines indicate the substructure shares by both compounds. 149 2,2’,4,4’-tetrachIorobiphenyl BEL Superposition Figure Vl.14. Superposition Of 2,2’,4,4’-tetrachlorobiphenyl and BEL, an inhibitor or Ca2*-independent PLA2. 150 compounds could be superimposed with 2,2’,4,4’-tetrachlorObiphenyl revealing the presence Of a motif characteristic to all Of them. We have called this substructure the OG motif. The OG motif was absent in the inactive OCs. External validation Of the model with some halogenated compounds revealed that the OG motif is present in the active compounds 2,4’- diclorobiphenyl, 2,2’-dibromobiphenyl and aldrin. However, it Is absent in the inactive compounds 4,4’-dichlorobiphenyl and 4,4’-dibromobiphenyl. VI. 5. Discussion In this chapter the use Of an empirical model tO classify chlorinated compounds as activators Of the neutrophil, BEL-sensitive PLA2 has been described. The results Of this SAR study suggest that OC molecules use a single, molecular substructure, the OG motif, tO activate the Ca2+- independent isoform Of PLAZ. It is tempting to summarize the SAR study in terms Of structural requirements that determine the activity Of OCs toward PLA2. Three factors are necessary to define the OG motif present in OC compounds. First is the presence Of a planar like-structure that is mainly hydrophobic (PH) and measures between 4.6 A (or-HCCH) and 7.6 A (BEL). The second is the presence Of an electronegative (ENR) region located perpendicular to one corner Of the planar structure within 1.8 A (OL- HCCH) tO 2.7 A (2,2’,4,4’-tetrachlorobiphenyl). The third part is a rigid 151 connector or bridge between PH and ENR. This bridge is important to generate some rigidity tO position the ENR in the binding site. This steric requirement may suggest the presence Of a protein receptor. A diagram Of the OG motif is shown in Figure Vl.15. It is important tO mention that one advantage Of this approach is that it considers simultaneously both topological and electronic features Of the molecule and does not require additional interpretation as from molecular descriptors Obtained from classical QSARs. SARs can provide good activity prediction for compounds in homologous series. Although the compounds presented here are Of diverse molecular structure, they may be considered as an homologous series, and that emphasizes the usefulness Of the model. This approach Of looking at sub-structures associated with toxicity Of OCs has been applied successfully to study the effects Of dieldrin in GABA channel functioning (Matsumura, 1985) and the assessment Of carcinogenicity by OC compounds (King and Srinivasan, 1996). The activation of PLAZ by the compounds having the OG motif would occur by assuming a model in which each compound interacts with a hydrophobic pocket within PLA2 and with an electropositive charge nearby. Given that orthO-chlorinated and not non-orthO-chlorinated PCBs activate PLA2, the steric rotational impediment Of orthO-PCBs may be necessary for their activity. It is consistent with the model that activation may depend on the possibility Of the 2-chlorine Of PCBs to interact with a positive site in the 152 Toxicophore for 0G motif Protein Figure Vl.15. Representation of the CG motif of OC compounds interacting with the toxicophore. 153 toxicophore. This toxicophore, presumably present in PLA2, is the critical local molecular fragment that is responsible for the activity Of OCs in neutrophils. OC compounds which are able tO penetrate cell membranes and interact with PLA2 are good probes to study the biochemical properties Of this enzyme. Direct binding studies are necessary to understand fully the mechanism of activation Of PLA2 by OCs. It is likely that OCs interact directly with the active site Of the PLA2 due to their molecular similarity to BEL. It is known that BEL binds covalently to the enzyme in the active site inhibiting its activity (Hazen et al., 19910). One shortcoming Of this SAR model is that is cannot provide information on the different potencies Of the compounds. Consequently, secondary parameters in addition to the OG motif, such as energy Of orbitals, charge density and number Of chlorines, are necessary for potency prediction. One interesting and important Observation from these studies is the possibility Of similar biological activity among OCs for which structural similarity is not Obvious from 2-D rendering. The molecular modelling data showed that DDT, dieldrin, chlordane, a-HCCH, 6—HCCH and y-HCCH, are likely tO behave as PCB like-compounds in some biochemical systems and vice-versa. 154 It should be stated that the activity predicted from SAR methods Is an estimate and that the geometry from quantum calculations may not be entirely correct. However, the concept that quantum chemical approaches can help to clarify mechanistic questions involving drug-receptor interactions has been reinforced (Randic, 1991). In addition, it has been suggested that SARs could and should be used in the hazard assessment process (Fiedler et al., 1990). Accordingly, the biochemical activity Of OC compounds having the OG motif should be considered when assessing potential toxicological effects. Based on this model we predict that other chemicals having the OG motif substructure such as toxaphene and in general all the orthO-PCBs and on‘hO-brominated biphenyls are likely to activate neutrophil PLA2. On the other hand, compounds such as polychlorinated naphthalenes, dioxins or other non-orthO-PCBs are not likely to activate PLA2. It may be concluded that organochlorine and organobromine compounds having the OG motif have a good likelihood to activate neutrophil PLA2 and consequently induce neutrophil activation to produce superoxide anion and release enzymes. 155 CHAPTER VII Summary 156 One Of the legacies Of the twentieth century is the creation Of new compounds probably unknown tO nature. Although we have managed to produce and use millions Of tons Of the new substances, we know little about their relationship with our living biochemistry. There is no doubt that the world as we see it today would have not been possible without the widespread use Of pesticides and related chemicals. Among the arsenal Of compounds developed this century, organochlorine (OC) compounds have proven important for the food industry, polymerization processes, military activities and human health. Compounds such as polychlorinated biphenyls, DDT, hexachlorocyclohexanes and dieldrin, among other organochlorines, were extensively used without the knowledge Of their potential toxicity and effects on the ecosystems. Many years later, their legal production has been halted or banned in many countries. However, these compounds remain accumulated in all the ecosystems and probably are permanently flowing in our own blood. Although during the last twenty years a great amount Of information on the toxicity Of these compounds has been compiled, there are still many effects which remain tO be understood. PCBs have been extensively studied and some Of their physiological targets identified. Alterations in the chemistry Of the central nervous 157 system, impairment Of neutrophil function, and cancer are some Of the effects attributed to these compounds. Polychlorinated biphenyls are defined as biphenyls having their hydrogens substituted by chlorines, theoretically producing 209 different congeners. It has been proposed that the presence or absence Of A chlorines in the 2-position Of one Of the phenyls is sufficient tO toxicologically classify these compounds. Accordingly, PCBs substituted on the 2-position (orthO-PCBs) have low affinity for the Ah receptor and are considered to have non-dioxin-Iike toxicity. On the other hand, the absence Of the chlorine in this position allows the compound to have dioxin-like toxicity with higher affinity for the Ah receptor. In the immune system, neutrophils have been the focus Of diverse studies including this one. PCBs cause activation Of NADPH oxidase and degranulation. The biochemical mechanisms leading tO these changes in neutrophil function include interference with signaling involving Ca2+, tyrosine kinases, phospholipase C and phospholipase A2 (PLAZ). The goal Of this thesis was tO test the hypothesis that in neutrophils PCBs cause changes in Ca2+ homeostasis and PLA2 by independent mechanisms and that PLA2 activation mechanisms can be triggered by any OC compound sharing a particular substructure similar tO that found in orthO-chlorinated PCBS. 158 The PCB mixture Aroclor 1242 and 2,2’,4,4’-tetrachlorobiphenyl induced increases in [Ca2+],. These effects were independent Of the activity Of PLA2 based on the lack Of effect Of BEL tO inhibit the Aroclor 1242-induced increase in [Ca2+]i. In addition, Aroclor 1242 blocked Ca” influx elicited by the bacterial product fMLP and failed to increase [CaZ+]i after intracellular store depletion by this chemotactic agent. Given that the changes in [C321, caused by fMLP may be important in the ability Of the neutrophil to defend against bacteria, these results suggest that the PCB mixture Aroclor 1242 may lead to neutrophil dysfunction in presence Of pathogens. Changes in the [Ca2+]i are generally associated with enzyme activation, particularly for those enzymes having Ca2+-binding domains. One Of these enzymes is cPLAz. PCBs, particularly on‘hO-PCBs, are known to target the neutrophil iPLAz; however, the Observation that PCBs also increase [Ca2+], was the first suggestion that more than one isoform Of PLA2 may be activated by PCBs. Inhibition Of TK, p42/p44 MAPK, or PKC decreased around 20-30% the PLAZ activity induced by Aroclor 1242. Similar results were Observed for the orthO-PCB 2,2’,4,4’-tetrachlorobiphenyl, which also showed sensitivity to inhibition Of ras farnesylation. These results suggest that a phosphorylation cascade involving TK, PKC, ras and MAPK regulate a fraction Of PCB-stimulated PLA2 activity. Currently, the only PLA2 known 159 to be activated by phosphorylation is cPLA2. Thus, these results suggest that in addition tO iPLA2, Aroclor 1242 may activate cPLAz. This suggestion was confirmed with the Observation that 2,2’,4,4’- tetrachlorobiphenyl induced phosphorylation Of cPLAz, In addition, this PCB also phosphorylates p44 MAPK, an upstream event in cPLA2 activation. Little is known about the regulation Of iPLA2. In some systems, this enzyme is present in a complex with calmodulin. Calmodulin inhibitors decreased Aroclor 1242-induced PLA2 activity. Interestingly, calmodulin inhibitors also blocked fMLP-induced neutrophil degranulation, as did Aroclor 1242. These results suggest that calmodulin-dependent processes are also involved in the activity Of PCBs in this system. The conceptual framework regarding the different signalling pathways involved in the activation Of neutrophils by PCB is summarized in Figure Vll.1. In the environment, PCBs are not found alone. In general, they are present with other organochlorine compounds such as DDT, dieldrin and chlordane among others. Some Of these compounds have been shown to activate neutrophils (Hewett and Roth, 1988). Despite their diversity in structure, we wondered if those compounds would not only activate PLA2 but also present some common molecular characteristics responsible for that activity. Molecular modeling techniques uncovered a common motif present in orthO-chlorinated PCBs, dieldrin, and OL-, orthO- 160 chlorinated PCBs and B-hexachlorocyclohexane. This motif has been called the OG motif. It has three parts, a planar hydrophobic structure, a . negatively charged atom perpendicular tO the plane and a rigid bridge connecting these two. The OG motif might be responsible for all the biological activities shared by orthO-PCBs and pesticides such as DDT, dieldrin and chlordane. Accordingly, this molecular characteristic can be the link between the effects Of PCBs in neutrophils and other cells, such as neurons where OIthO-PCBs have shown similar biological properties. These results demonstrate the utility Of molecular modeling for identifying substructures with potential toxicity, and open a door tO better understand the relationships between molecular recognition and biological activity for organochlorine compounds. 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